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WO2025034518A1 - Instrumentation et procédés d'analyse d'acides nucléiques - Google Patents

Instrumentation et procédés d'analyse d'acides nucléiques Download PDF

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Publication number
WO2025034518A1
WO2025034518A1 PCT/US2024/040578 US2024040578W WO2025034518A1 WO 2025034518 A1 WO2025034518 A1 WO 2025034518A1 US 2024040578 W US2024040578 W US 2024040578W WO 2025034518 A1 WO2025034518 A1 WO 2025034518A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
cases
reporter
fold
nucleotides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/040578
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English (en)
Inventor
Timothy James PATNO
Phillip You Fai Lee
Devin SPRATT
Nathan WRIGHT
Alex BORISEVICH
Thomas Atkinson
Mazen KHABBAZ
Lisa J. KRYGSMAN
Lars Gustafson
Janice Sha CHEN
Alistair WARD
Harry Turner
Nicholas John Collier
Mark Ridley
Alan DOLBY
Alex BARKER
Euan Morrison
Roger Millington
Daniel Thomas DRZAL
James Gani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mammoth Biosciences Inc
Original Assignee
Mammoth Biosciences Inc
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Application filed by Mammoth Biosciences Inc filed Critical Mammoth Biosciences Inc
Publication of WO2025034518A1 publication Critical patent/WO2025034518A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1861—Means for temperature control using radiation
    • B01L2300/1872—Infrared light
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00039—Transport arrangements specific to flat sample substrates, e.g. pusher blade
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks

Definitions

  • Nucleic acid tests can be used to detect and identify nucleic acid sequences associated with disease-causing organisms, such as influenza. Diagnostic tests that detect the presence or absence of disease-associated nucleic acids often have an advantage in sensitivity and specificity over other test methods, such as those based on protein detection (e.g., antigens of a pathogenic organism).
  • protein detection e.g., antigens of a pathogenic organism.
  • existing lower cost, easy-to-use nucleic acid-based test systems are limited in their ability to perform multiplexing due at least in part to difficulties in signal differentiation while trying to maintain a high reliability at lower cost and greater ease of use.
  • the present disclosure provides cartridges and devices for detecting nucleic acids, systems comprising the same, and methods of use thereof.
  • the systems or components thereof may be configured for one or more of initial sample processing (e.g., nucleic acid extraction), nucleic acid amplification, and/or nucleic acid detection.
  • the system or a component thereof e.g., a cartridge
  • the system or a component thereof is configured for nucleic acid amplification.
  • the system or a component thereof is configured for nucleic acid amplification and detection.
  • a non-limiting example of a nucleic acid detection comprises methodologies using a programmable nuclease.
  • An aspect of the present disclosure provides a system for detecting a target nucleic acid, the system comprising: an instrument configured to interface with a cartridge; wherein: (a) the instrument includes: (i) one or more pumps; (ii) at least one valve actuator; (iii) a light source configured to illuminate a detection region of the cartridge; and (iv) an optical sensor configured to detect one or more signals from the detection region; (b) the cartridge includes: (i) a sample interface configured to receive a sample including one or more nucleic acids; (ii) one or more reagent capsules; (iii) a sample preparation region; (iv) a detection region; (iv) and a plurality of pump interfaces fluidically connected to the sample interface, the one or more reagent capsules, the sample preparation region, and the detection region via a plurality of valves; (c) the one or more pumps is configured to apply positive and/or negative pressure through the plurality of pump interfaces; (d) the at least one valve actuator
  • each chamber of the plurality of chambers further includes detection reagents including a guide nucleic acid and a reporter, and further wherein: (a) each guide nucleic acid (i) includes a targeting sequence that hybridizes with a target nucleic acid of a plurality of different target nucleic acids or an amplicon thereof, and (ii) is effective to form a complex with a programmable nuclease that is activated upon binding the corresponding target nucleic acid or amplicon thereof; (b) the guide nucleic acid of a first chamber in the plurality of chambers includes a different targeting sequence from the guide nucleic acid of a second chamber in the plurality of chambers; and (c) each reporter (i) includes a cleavable nucleic acid and a detection moiety, and (ii) is configured to be cleaved to form a detectable cleavage product in response to activation of the complex in the respective chamber.
  • the one or more reagent capsules comprises dried reagents;
  • the cartridge comprises one or more fluid reservoirs; and
  • a pump of the one or more pumps is configured to move fluid from the fluid reservoir to the one or more reagent capsules.
  • the instrument comprises one or more heaters; and (b) at least one of the one or more heaters is configured to interface with the detection region and heat contents thereof.
  • the sample preparation region comprises a lysis region for the lysis one or more components of the sample; and optionally (b) at least one of the one or more reagent capsules comprises lysis reagents.
  • At least one of the one or more heaters is configured to interface with the sample preparation region and heat the contents thereof.
  • the cartridge further comprises a nucleic acid capture region;
  • the one or more pumps comprises an actuator operable to move a sample preparation fluid through the nucleic acid capture region to capture the one or more nucleic acids; and
  • the one or more pumps comprises an actuator operable to move an elution fluid from one of the one or more fluid reservoirs through the nucleic acid capture region to release the captured nucleic acids.
  • At least one of the one or more reagent capsules or the plurality of chambers comprises a programmable nuclease.
  • the programmable nuclease comprises a Cas protein; and optionally (b) the Cas protein comprises Casl2, Casl3, Cast 4, CasPhi, a thermostable Cas, or any combination thereof.
  • the detection reagents further comprise amplification reagents; and optionally (b) the amplification reagents in each chamber of the plurality of chambers comprises one or more primers for amplifying the target nucleic acid bound by the respective targeting sequence.
  • the detection reagents are in a lyophilized form, and/or (b) the guide nucleic acid and/or the reporter in each chamber are immobilized to a surface of the respective chamber.
  • the system further comprises a gear that translates the cartridge between two or more positions in the instrument.
  • the two or more positions comprise positions in which (a) one of the one or more pumps aligns with one or more of the plurality of pump interfaces; (b) one of the at least one valve actuator aligns with one or more of the plurality of valves; (c) the optical sensor aligns with the detection region; or (d) any combination thereof.
  • the two or more positions comprise a position in which at least one of the one or more heaters aligns with the sample preparation region or the detection region.
  • the system further comprises a user interface for receiving instructions from a user.
  • the system further comprises a non-transitory computer-readable medium with instructions stored thereon, that when executed by one or more processors, (a) activates the one or more pumps and the at least one valve actuator to move fluid between the sample interface, the one or more reagent capsules, the sample preparation region, and the detection region; (b) activates the light source; (c) stores detection results from the optical sensor; and (d) reports results to a user.
  • the present disclosure provides methods for detecting one or more of a plurality of different target nucleic acids.
  • the detecting is performed using any of the systems described herein.
  • the method comprises: (a) flowing a liquid comprising one or more of the different target nucleic acids or amplicons thereof into the plurality of chambers; (b) in one or more of the plurality of chambers, forming the activated complex and cleaving the reporters; and (c) detecting the detectable cleavage products in one or more of the plurality of chambers, wherein the location of a chamber comprising a detectable cleavage product identifies the target nucleic acid or amplicon thereof present in the chamber.
  • FIGS. 1A-1C show representations of exemplary cartridge embodiments and instrument structure.
  • FIG. 1A and FIG. 1C show representations of exemplary cartridges described herein.
  • FIG. IB shows a representation of an exemplary instrument for use with cartridges described herein. Illustrative dimensions are shown in inches.
  • FIGS. 2A-2C show illustrative views and schematic representations of a cartridge and operations thereof, according to some embodiments.
  • FIG. 2A shows a schematic representation of a process workflow.
  • FIG. 2B shows a representation of an exemplary cartridge (top view). Elements illustrated in FIG. 2B include dried reagents (enzyme (E), activator (A), extra reagent (ER), proteinase K (PK), and lysis (L)), vents (v), and pumps to transfer/mix (P).
  • FIG. 2C shows a representation of an exemplary cartridge (perspective view).
  • FIG. 3 shows a schematic representation of a process workflow in a cartridge of FIGS. 2A-2C, with components used during a sample lysis step indicated by outline.
  • FIG. 4 shows a schematic representation of a process workflow in a cartridge of FIGS. 2A-2C, with components used during a nucleic acid binding/capture step indicated by outline.
  • FIG. 5 shows a schematic representation of a process workflow in a cartridge of FIGS. 2A-2C, with components used during one or more washing steps indicated by an outline.
  • FIG. 6 shows a schematic representation of a process workflow in a cartridge of FIGS. 2A-2C, with components used during an elution step and a detection step indicated by an outline.
  • FIG. 7 shows an illustrative schematic representation for the operation of a two-pump, two-valve cartridge, in accordance with some embodiments.
  • Illustrated elements include dried tRNA (R), dried lysis reagent (L), neutralizer (N), activator (A), and LAMP + CRISPR reagents (L/C).
  • FIGS. 8A-8B show side (FIG. 8A) and top (FIG. 8B) view representations of an exemplary cartridge housing, including dimensions indicated in millimeters and inches.
  • FIG. 9 shows a perspective view representation of an exemplary cartridge housing.
  • FIGS. 10A-10B shows views of internal components of an exemplary cartridge with housing removed, according to some embodiments.
  • FIG. 10A shows a perspective view of the cartridge.
  • FIG. 10B shows: (a) a perspective view of a dry reagent capsule body, perspective view with transparent body housing, and cross-sectional view along YZ plane of the dry reagent capsule body (top row, left to right); and (b) an exploded view of a dry reagent capsule assembly, and cross-sectional view of assembled dry reagent capsule along YZ plane including connections to fluid channels below the dry reagent capsule (bottom row, left to right).
  • FIG. 10C shows an exploded view of an exemplary cartridge assembly, in accordance with embodiments.
  • FIG. 11 shows an exploded view of a cartridge rotary valve, according to some embodiments.
  • Part A includes the fluid holes that act as the valve inlet and outlet ports.
  • Part B is an elastomeric sealing gasket that prevents leaking.
  • Part B is affixed to Part C by over-molding adhesion of the Part B and Part C plastics.
  • Part D compresses the combined Parts B and C to Part A.
  • Part D is joined to Part A by laser welding or ultrasonic welding or the like.
  • the joining of Part D to Part A also forms fluid paths on the cartridge bottom.
  • a similar joining of another plastic part to the top of Part A also provides fluidic chambers and channels (now shown).
  • FIG. 12 shows a perspective view representation of dry reagent chambers of an exemplary cartridge and a detection region (e.g., for a DETECTR reaction).
  • FIG. 13 shows a layout diagram of an exemplary detection region (e.g., for a DETECTR reaction).
  • FIG. 14 shows cartridge positions in various zones within an exemplary instrument described herein.
  • FIG. 15 shows cartridge working zones with optical interfaces highlighted.
  • FIG. 16 is an illustration of an exemplary instrument (with housing and display removed) comprising four instrument modules shown that directly interface with a cartridge as it is moved through the exemplary instrument by a cartridge mover module.
  • FIG. 17 is an illustration of an exemplary instrument showing an exemplary cartridge for insertion into an instrument described herein.
  • FIG. 18 is an illustration of an isometric frontal view of an exemplary instrument showing an exemplary cartridge in an instrument described herein.
  • FIG. 19 is an illustration of an instrument module contacting an exemplary cartridge.
  • FIG. 20 is an illustration of a cartridge mover module, with a gear that translates a cartridge between two or more positions in an instrument described herein.
  • FIG. 21 is an illustration of a layout of optical components in an instrument’s optical module, in accordance with some embodiments.
  • FIG. 22 is an illustration of an exemplary thermal module for heating portions of a cartridge described herein.
  • FIG. 23 is an exploded view of an exemplary thermal module, according to some embodiments.
  • FIG. 24 is an illustration of an isometric view of a thermal module in an instrument described herein.
  • FIG. 25 is an illustration of a pump module including reagent capsule pushers and actuators, according to some embodiments.
  • FIG. 26 is an illustration of a pump module viewed from within the cartridge housing, according to some embodiments.
  • FIG. 27 is a side view illustration of a cartridge interfacing with a pump module and rotary valve actuator, according to some embodiments.
  • FIG. 28 is an illustration of a rotary valve actuator, according to some embodiments.
  • FIGS. 29A-29B are illustrations of a rotary valve actuator contacting a cartridge, according to some embodiments.
  • FIG. 29A is an illustration of an external view of a rotary valve actuator contacting a cartridge.
  • FIG. 29B is an illustration of a perspective view of a rotary valve actuator showing where it contacts a cartridge, with transparent cartridge for ease of viewing.
  • FIG. 30 is an illustration of a cartridge bottom with three rotary valves oriented for instrument loading and unloading, in accordance with some embodiments.
  • FIG. 31 is an illustration of an instrument back without a cover, showing power and communications connection at the bottom.
  • FIG. 32 is an exploded view of an instrument showing communications and controller modules, according to some embodiments.
  • FIGS. 33A-33B depict representations of illustrative microfluidic devices comprising a plurality of chambers fluidically connected in sequence, corresponding to a detection region of a cartridge in accordance with some embodiments.
  • Each of the illustrated chambers comprises a well, an inlet channel, an outlet, and a capillary valve.
  • the locations of wells, capillary valves, and outlets are labeled.
  • FIGS. 33A and 33B present alternative arrangements of the capillary valves and outlets relative to the respective wells.
  • an outlet defines a volume of the respective chamber that is separate from but fluidically connected to the respective well.
  • an outlet corresponds to an opening of the respective well, which may optionally be sealed by an air-permeable membrane (not shown).
  • FIGS. 34A-34C depict different views of a microfluidic device in accordance with some embodiments.
  • FIG. 34A shows a perspective view of a top surface of a detection region of the microfluidic device.
  • FIG. 34B shows a perspective cross-sectional view of the top surface of the detection region.
  • FIG. 34C shows a plan view of the top surface of the detection region.
  • the illustrated microfluidic device comprises a plurality of chambers fluidically connected in sequence, in which each chamber of the plurality of chambers comprises a well, an inlet channel, an outlet, and a capillary valve.
  • the arrangement of features in the device and fluid flow within the device is similar to those illustrated in FIG. 33A.
  • FIG. 35 depicts a perspective view of a bottom surface of the detection region of the microfluidic device shown in FIGS. 34A-34C. Optional features added to the bottom of the device around the outlets to help ultrasonically weld an air-permeable membrane to the device are shown.
  • FIG. 36 depicts a perspective view of a top surface of a microfluidic device in accordance with some embodiments.
  • the illustrated device is similar to that illustrated in FIGS. 34A-34C, except a film is used instead of a molded lid.
  • FIG. 37 depicts a perspective cross-sectional view of a microfluidic device in accordance with some embodiments.
  • the illustrated device is similar to that illustrated in FIGS. 34A-34C, except the membrane can be compounded with an adhesive backing to adhere to the device instead of using laser welding or ultrasonic welding or heat staking.
  • FIGS. 38A-38D depict an exemplary detection region of a microfluidic device in accordance with some embodiments.
  • FIGS. 38A-38B show perspective and plan views of the top of the device, respectively.
  • FIG. 38C-38D show perspective and plan views of the bottom of the device, respectively.
  • the illustrated device includes a plurality of chambers fluidically connected in sequence, and connected to an upstream first inlet channel by way of a bubble purge channel (or a bubble trap) disposed therebetween. In operation, bubbles in the liquid of the first inlet channel enter the bubble purge channel and escape through a hydrophobic or oleophobic membrane covering the bottom surface of the bubble purge channel.
  • the liquid itself is substantially prevented from passing through the membrane and instead continues to flow along the bubble purge channel towards its exit into a second inlet channel, reduced in or substantially free of bubbles.
  • the second inlet channel feeds into the plurality of chambers. Also illustrated is the location of the hydrophobic and/or oleophobic membrane that covers at least a portion of a bottom surface of the device.
  • FIG. 39 depicts a surface comprising an immobilized programmable nuclease-guide nucleic acid complex and a plurality of reporters, where one reporter has been cleaved by an activated programmable nuclease, in accordance with embodiments.
  • FIGS. 40A-40E depict exemplary reporters immobilized on a substrate, in accordance with embodiments. Arrows indicate signal (e.g., fluorescence) change when the reporter is cleaved by a programmable nuclease.
  • signal e.g., fluorescence
  • FIG. 41 depicts an exemplary high-level workflow for using a multiplex detection system, in accordance with some embodiments.
  • the illustrated process provides a multiplex method for detecting target nucleic acids from influenza virus A (IVA), influenza virus B (IVB), respiratory syncytial virus (RSV), SARS-CoV-2 (SC2), and RNase P (RP).
  • the illustrated reaction containers correspond to reaction locations withing a cartridge described herein (e.g., lysis, mixing, and amplification/detection regions).
  • FIG. 42 depicts an exemplary sample collection workflow, in accordance with embodiments.
  • structures, systems, and/or devices described herein may be embodied as integrated components or as separate components.
  • the present disclosure is described in relation to systems, devices, or methods for in vitro diagnostics, and in particular for detection of an ailment such as a disease, cancer, or genetic disorder.
  • an ailment such as a disease, cancer, or genetic disorder.
  • the devices and methods disclosed herein may be used in other nucleic acid testing including, but not limited to, detecting genetic information, such as for phenotyping, genotyping, determining ancestry, or the like.
  • the present disclosure provides systems and methods for nucleic acid target detection.
  • the systems and methods of the present disclosure can be implemented using devices that are configured for programmable nuclease-based detection.
  • the devices can be configured for single reaction detection.
  • the devices can be configured for multiplexed detection.
  • the target can comprise a target sequence or target nucleic acid.
  • a target can be referred to interchangeably as a target nucleic acid.
  • a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling or isothermal process as described elsewhere herein).
  • the target nucleic acid or amplicon thereof can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any plant, animal, virus, or microbe of interest.
  • the devices provided herein can be used to perform rapid tests in a single integrated system.
  • one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule).
  • a programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter and generation of a signal, directly or indirectly, therefrom.
  • cleavage of a reporter by a programmable nuclease may release a detection moiety which generates a detectable signal when cleaved from the reporter.
  • the signal may be an increase in a signal (e.g., an increase in fluorescence upon release of a quencher as described herein), a decrease in signal (e.g., a decrease in fluorescence upon release of a fluorophore as described herein), or any other change in signal (e.g., a color change) as will be understood by one of ordinary skill in the art.
  • the detection moiety may trigger a downstream signal amplification reaction (e.g., the detection moiety may comprise an enzyme which, upon release from the reporter, can contact its substrate and result in a detectable color change) which can increase the amount of detectable signal generated per reporter cleavage event.
  • the present disclosure provides systems and methods for target nucleic acid detection.
  • the systems and methods of the present disclosure can be implemented using devices that are configured for programmable nuclease-based detection.
  • the devices can be configured for single reaction detection.
  • the devices can be disposable devices.
  • the devices disclosed herein can be particularly well-suited for carrying out highly efficient, rapid, and accurate reactions for detecting whether a target is present in a sample.
  • the target can comprise a target sequence or target nucleic acid.
  • a target can be referred to interchangeably as a target nucleic acid.
  • a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling or isothermal process as described elsewhere herein).
  • the target nucleic acid can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any plant, animal, virus, or microbe of interest.
  • the devices provided herein can be used to perform rapid tests in a single integrated system.
  • the target nucleic acid can be a nucleic acid or a portion of a nucleic acid from a pathogen, virus, bacterium, fungi, protozoa, worm, or other agent(s) or organism(s) responsible for and/or related to a disease or condition in living organisms (e.g., humans, animals, plants, crops, and the like).
  • the target nucleic acid can be a nucleic acid, or a portion thereof.
  • the target nucleic acid can be a portion of a nucleic acid from a gene expressed in a cancer or genetic disorder in the sample.
  • the target nucleic acid can be a portion of an RNA or DNA from any organism in the sample.
  • the sample can be used for identifying a disease status or condition.
  • a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject.
  • a method comprises obtaining a serum sample from a subject and identifying a disease status or condition of the subject.
  • a method comprises obtaining a nasal swab from a subject and identifying a disease status or condition of the subject.
  • the sample comprises a target nucleic acid.
  • the sample comprises a plurality of target nucleic acids.
  • the target nucleic acid is a single stranded nucleic acid.
  • the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents.
  • the target nucleic acid may be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples.
  • the target nucleic acids include but are not limited to mRNA, rRNA, tRNA, noncoding RNA, long non-coding RNA, and microRNA (miRNA).
  • the target nucleic acid is mRNA.
  • the target nucleic acid is from a virus, a parasite, or a bacterium described herein.
  • the target nucleic acid is transcribed from a gene as described herein.
  • a number of target nucleic acids are consistent with the methods and compositions disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least two copies of the target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acid copies.
  • the method detects target nucleic acid present at least at one copy per 10 1 non-target nucleic acids, 10 2 non-target nucleic acids, 10 3 non-target nucleic acids, 10 4 non-target nucleic acids, 10 5 nontarget nucleic acids, 10 6 non-target nucleic acids, 10 7 non-target nucleic acids, 10 8 non-target nucleic acids, 10 9 non-target nucleic acids, or 10 10 non-target nucleic acids.
  • the systems and methods of the present disclosure can be used to detect one or more target sequences or nucleic acids in one or more samples.
  • the one or more samples can comprise one or more target sequences or nucleic acids for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein.
  • a sample can be taken from any place where a nucleic acid can be found.
  • Samples can be taken from an individual/human, a non-human animal, or a crop, or an environmental sample can be obtained to test for presence of a disease, virus, pathogen, cancer, genetic disorder, or any mutation or pathogen of interest.
  • a biological sample can be blood, serum, plasma, lung fluid, exhaled breath condensate, saliva, spit, urine, stool, feces, mucus, lymph fluid, peritoneal , cerebrospinal fluid, amniotic fluid, breast milk, gastric secretions, bodily discharges, secretions from ulcers, pus, nasal secretions, sputum, pharyngeal exudates, urethral secretions/mucus, vaginal secretions/mucus, anal secretion/mucus, semen, tears, an exudate, an effusion, tissue fluid, interstitial fluid (e.g., tumor interstitial fluid), cyst fluid, tissue, or, in some instances, any combination thereof.
  • tissue fluid interstitial
  • a sample can be an aspirate of a bodily fluid from an animal (e.g., human, animals, livestock, pet, etc.) or plant.
  • a tissue sample can be from any tissue that can be infected or affected by a pathogen (e.g., a wart, lung tissue, skin tissue, and the like).
  • a tissue sample (e.g., from animals, plants, or humans) can be dissociated or liquified prior to application to detection system of the present disclosure.
  • a sample can be from a plant (e.g., a crop, a hydroponically grown crop or plant, and/or house plant). Plant samples can include extracellular fluid, from tissue (e.g., root, leaves, stem, trunk etc.).
  • a sample can be taken from the environment immediately surrounding a plant, such as hydroponic fluid/ water, or soil.
  • a sample from an environment can be from soil, air, or water.
  • the environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest.
  • the raw sample is applied to the detection system.
  • the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system.
  • the sample is contained in no more than about 200 nanoliters (nL). In some cases, the sample is contained in about 200 nL. In some cases, the sample is contained in a volume that is greater than about 200 nL and less than about 20 microliters (pL).
  • the sample is contained in no more than 20 pl. In some cases, the sample is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 pl, or any of value from 1 pl to 500 pl.
  • the sample is contained in from 1 pL to 500 pL, from 10 pL to 500 pL, from 50 pL to 500 pL, from 100 pL to 500 pL, from 200 pL to 500 pL, from 300 pL to 500 pL, from 400 pL to 500 pL, from 1 pL to 200 pL, from 10 pL to 200 pL, from 50 pL to 200 pL, from 100 pL to 200 pL, from 1 pL to 100 pL, from 10 pL to 100 pL, from 50 pL to 100 pL, from 1 pL to 50 pL, from 10 pL to 50 pL, from 1 pL to 20 pL, from 10 pL to 20 pL, or from 1 pL to 10 pL. Sometimes, the sample is contained in more than 500 pl.
  • the sample is taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine.
  • the sample is taken from nematodes, protozoans, helminths, or malarial parasites.
  • the sample may comprise nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell.
  • the sample may comprise nucleic acids expressed from a cell.
  • the sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein.
  • the target sequence is a portion of a nucleic acid.
  • a nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA.
  • a nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length.
  • a nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length.
  • a nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from 35 to 50, or from 45 to 50 nucleotides in length.
  • a nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • the target nucleic acid can be reverse complementary to a guide nucleic acid. In some cases, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
  • nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.
  • the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample.
  • the target sequence in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample.
  • the target sequence in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample.
  • the target sequence in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample.
  • the target sequence is a portion of a nucleic acid from sepsis, in the sample.
  • diseases can include but are not limited to respiratory viruses (e.g., SARS-CoV-2 (i.e., a virus that causes COVID-19), SARS, MERS, influenza, Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Metapneumovirus (hMPV), Human Rhinovirus/Enterovirus, Influenza A, Influenza A/Hl, Influenza A/H3, Influenza A/Hl-2009, Influenza B, Influenza C, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus) and respiratory bacteria (e.g.
  • respiratory viruses e.g., SARS-CoV-2 (i.e., a virus that causes
  • Bordetella parapertussis Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae).
  • Other viruses include human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis.
  • Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites.
  • Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms.
  • Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.
  • pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii.
  • Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, and Candida albicans.
  • Pathogenic viruses include but are not limited to: respiratory viruses (e.g., adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, MERS), gastrointestinal viruses (e.g., noroviruses, rotaviruses, some adenoviruses, astroviruses), exanthematous viruses (e.g., the virus that causes measles, the virus that causes rubella, the virus that causes chickenpox/shingles, the virus that causes roseola, the virus that causes smallpox, the virus that causes fifth disease, chikungunya virus infection); hepatic viral diseases (e.g., hepatitis A, B, C, D, E); cutaneous viral diseases (e.g., warts (including genital, anal), herpes (including oral, genital, anal), molluscum contagiosum); hemmorhagic viral diseases (e.g.
  • respiratory viruses e.g.
  • Ebola Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever
  • neurologic viruses e.g., polio, viral meningitis, viral encephalitis, rabies
  • sexually transmitted viruses e.g., HIV, HPV, and the like
  • immunodeficiency virus e.g., HIV
  • influenza virus dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like.
  • Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus anthracis, Bortadella pertussis, Burkholderia cepacia, Corynebacterium diphtheriae, Coxiella burnetii, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella longbeachae, Legionella pneumophila, Leptospira interrogans, Moraxella catarrhalis, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria elongate, Neisseria gonorrhoeae, Parechovirus, Pneumococcus, Pneumocystis jirovecii, Crypto
  • the target nucleic acid may comprise a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample.
  • the target nucleic acid is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis.
  • HCV human immunodeficiency virus
  • HPV human papillomavirus
  • chlamydia gonorrhea
  • syphilis syphilis
  • trichomoniasis sexually transmitted infection
  • malaria Dengue fever
  • Ebola chikungunya
  • leishmaniasis leishmaniasis
  • Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites.
  • Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms.
  • Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.
  • pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii.
  • Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
  • Pathogenic viruses include but are not limited to immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like.
  • immunodeficiency virus e.g., HIV
  • influenza virus dengue; West Nile virus
  • herpes virus yellow fever virus
  • Hepatitis Virus C Hepatitis Virus A
  • Hepatitis Virus B Hepatitis Virus B
  • papillomavirus papillomavirus
  • Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Streptococcus pyogenes, Streptococcus salivarius, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory
  • the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.
  • the sample used for cancer testing or cancer risk testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
  • the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene with a mutation associated with cancer, from a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle.
  • the target nucleic acid encodes for a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer.
  • the assay can be used to detect “hotspots” in target nucleic acids that can be predictive of cancer, such as lung cancer, cervical cancer, in some cases, the cancer can be a cancer that is caused by a virus.
  • viruses that cause cancers in humans include Epstein-Barr virus (e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma); papillomavirus (e.g., cervical carcinoma, anal carcinoma, oropharyngeal carcinoma, penile carcinoma); hepatitis B and C viruses (e.g., hepatocellular carcinoma); human adult T-cell leukemia virus type 1 (HTLV-1) (e.g., T-cell leukemia); and Merkel cell polyomavirus (e.g., Merkel cell carcinoma).
  • Epstein-Barr virus e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma
  • the target nucleic acid is a portion of a nucleic acid that is associated with a blood fever.
  • the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICER1, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREM1, H0XB13, HRAS, KRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2,
  • the sample used for genetic disorder testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
  • the genetic disorder is hemophilia, sickle cell anemia, P-thalassemia, Duchene muscular dystrophy, severe combined immunodeficiency, or cystic fibrosis.
  • the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder.
  • the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: CFTR, FMRI, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, AC ADM, ACADVL, ACAT1, ACOX1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE, ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL, ASNS, ASP A, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290, CERKL
  • the sample used for phenotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
  • the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene associated with a phenotypic trait.
  • the sample used for genotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
  • the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene associated with a genotype.
  • the sample used for ancestral testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
  • the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene associated with a geographic region of origin or ethnic group.
  • the sample can be used for identifying a disease status.
  • a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject.
  • the disease can be a cancer or genetic disorder.
  • a method may comprise obtaining a serum sample from a subject; and identifying a disease status of the subject.
  • the disease status is prostate disease status.
  • the device can be configured for asymptomatic, pre-symptomatic, and/or symptomatic diagnostic applications, irrespective of immunity.
  • the device can be configured to perform one or more serological assays on a sample (e.g., a sample comprising blood).
  • the sample can be used to identify a mutation in a target nucleic acid of a plant or of a bacteria, virus, or microbe associated with a plant or soil.
  • the devices and methods of the present disclosure can be used to identify a mutation of a target nucleic acid that affects the expression of a gene.
  • a mutation that affects the expression of gene can be a mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene.
  • the mutation is a single nucleotide mutation.
  • the target nucleic acid is a single stranded nucleic acid.
  • the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents.
  • the target nucleic acid can be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples.
  • the target nucleic acids include but are not limited to mRNA, rRNA, tRNA, noncoding RNA, long non-coding RNA, and microRNA (miRNA).
  • the target nucleic acid is mRNA.
  • the target nucleic acid is from a virus, a parasite, or a bacterium described herein.
  • the target nucleic acid is transcribed from a gene as described herein.
  • a number of target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population.
  • the sample has at least 2 target nucleic acids.
  • the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids.
  • the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids.
  • the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to 6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to
  • the method detects target nucleic acid present at least at one copy per 10 1 non-target nucleic acids,
  • 10 2 non-target nucleic acids 10 3 non-target nucleic acids, 10 4 non-target nucleic acids, 10 5 nontarget nucleic acids, 10 6 non-target nucleic acids, 10 7 non-target nucleic acids, 10 8 non-target nucleic acids, 10 9 non-target nucleic acids, or 10 10 non-target nucleic acids.
  • a number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 different target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations.
  • the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations.
  • the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 10 1 non-target nucleic acids, 10 2 non-target nucleic acids,
  • the target nucleic acid populations can be present at different concentrations or amounts in the sample.
  • the target nucleic acid is indicative of a respiratory disorder or respiratory pathogen.
  • the respiratory disorder or respiratory pathogen selected from the group consisting of SARS-CoV-2 and corresponding variants, 29E, NL63, OC43, HKU1, MERS-CoV, (MERS), SARS-CoV (SARS), Flu A, Flu B, RSV, Rhinovirus, Strep A, and TB.
  • the device is configured to differentiate between a viral infection and a bacterial infection.
  • the target nucleic acid is indicative of a sexually transmitted infection (STI) or infection related to a woman’s health.
  • STI sexually transmitted infection
  • the STI or infection related to a woman’s health is selected from the group consisting of CT, NG, MG, TV, HPV, Candida, B. Vaginosis Syphilis, and UTI.
  • the target nucleic acid comprises a single nucleotide polymorphism (SNP).
  • the SNP is indicative of NASH disorder or Alpha-1 disorder.
  • the target nucleic acid is a blood borne pathogen selected from the group consisted of HIV, HBV, HCV, and Zika.
  • the target nucleic acid is indicative of H. Pylori, C. Difficile, Norovirus, HSV, and Meningitis.
  • programmable nucleases and uses thereof, e.g., detection and editing of target nucleic acids.
  • a programmable nuclease is capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment.
  • a programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence.
  • the programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and can non-specifically degrade a non-target nucleic acid in its environment.
  • the programmable nuclease has trans cleavage activity once activated.
  • a programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease or Cas effector protein).
  • a guide nucleic acid (e.g., crRNA) and Cas protein can form a CRISPR enzyme (also referred to herein as a programmable nuclease complex or probe).
  • one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule).
  • a programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter.
  • the programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease.
  • the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter.
  • a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease.
  • a programmable nuclease as described herein can be activated by a target RNA or a target DNA.
  • a programmable nuclease e.g., a Cas enzyme
  • the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters.
  • a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA- activated programmable DNA nuclease.
  • the nucleic acids described and referred to herein can comprise a plurality of base pairs.
  • a base pair can be a biological unit comprising two nucleobases bound to each other by hydrogen bonds.
  • Nucleobases can comprise adenine, guanine, cytosine, thymine, and/or uracil.
  • the nucleic acids described and referred to herein can comprise different base pairs.
  • the nucleic acids described and referred to herein can comprise one or more modified base pairs. The one or more modified base pairs can be produced when one or more base pairs undergo a chemical modification leading to new bases.
  • the one or more modified base pairs can be, for example, Hypoxanthine, Inosine, Xanthine, Xanthosine, 7-Methylguanine, 7-Methylguanosine, 5,6-Dihydrouracil, Dihydrouridine, 5-Methylcytosine, 5-Methylcytidine, 5- hydroxymethylcytosine (5hmC), 5 -formylcytosine (5fC), or 5-carboxylcytosine (5caC).
  • the programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity.
  • Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of reporter nucleic acids comprising a detection moiety.
  • the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal.
  • the reporter and/or the detection moiety can be immobilized, dried, or otherwise deposited on a support medium.
  • the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid.
  • the detection moiety binds to a capture molecule on the support medium to be immobilized.
  • the detectable signal can be visualized on the support medium to assess the presence or concentration of one or more target nucleic acids associated with an ailment, such as a disease, cancer, or genetic disorder.
  • the systems and methods of the present disclosure can be implemented using a device that is compatible with any type of programmable nuclease that is human-engineered or naturally occurring.
  • the programmable nuclease can comprise a nuclease that is capable of being activated when complexed with a guide nucleic acid and a target nucleic acid segment or a portion thereof.
  • a programmable nuclease can become activated when complexed with a guide nucleic acid and a target sequence of a target gene of interest.
  • the programmable nuclease can be activated upon binding of a guide nucleic acid to a target nucleic acid and can exhibit or enable trans cleavage activity once activated.
  • the systems and methods of the present disclosure can be implemented using a device that is compatible with a plurality of programmable nucleases.
  • the device can comprise a plurality of programmable nuclease probes (also referred to herein as programmable nuclease complexes) comprising the plurality of programmable nucleases and one or more corresponding guide nucleic acids.
  • the plurality of programmable nuclease probes can be the same.
  • the plurality of programmable nuclease probes can be different.
  • the plurality of programmable nuclease probes can comprise different programmable nucleases and/or different guide nucleic acids associated with the programmable nucleases.
  • a programmable nuclease generally refers to any enzyme that can cleave nucleic acid.
  • the programmable nuclease can be any enzyme that can be or has been designed, modified, or engineered by human contribution so that the enzyme targets or cleaves the nucleic acid in a sequence-specific manner.
  • Programmable nucleases can include, for example, zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and/or RNA- guided nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) nucleases or Cpfl.
  • Programmable nucleases can also include, for example, PfAgo and/or NgAgo.
  • ZFNs can cut genetic material in a sequence- specific matter and can be designed, or programmed, to target specific viral targets.
  • a ZFN is composed of two domains: a DNA- binding zinc-finger protein linked to the Fokl nuclease domain.
  • the DNA-binding zinc-finger protein is fused with the non-specific Fokl cleave domain to create ZFNs.
  • the protein will typically dimerize for activity.
  • Two ZFN monomers form an active nuclease; each monomer binds to adjacent half- sites on the target.
  • the sequence specificity of ZFNs is determined by ZFPs.
  • Each zinc-finger recognizes a 3 -bp DNA sequence, and 3-6 zinc-fingers are used to generate a single ZFN subunit that binds to DNA sequences of 9-18 bp.
  • the DNA-binding specificities of zinc-fingers is altered by mutagenesis.
  • New ZFPs are programmed by modular assembly of pre-characterized zinc fingers.
  • Transcription activator-like effector nucleases can cut genetic material in a sequence-specific matter and can be designed, or programmed, to target specific viral targets.
  • TALENs contain the Fokl nuclease domain at their carboxyl termini and a class of DNA binding domains known as transcription activator- like effectors (TALEs).
  • TALEs transcription activator- like effectors
  • TALENs are composed of tandem arrays of 33-35 amino acid repeats, each of which recognizes a single base-pair in the major groove of target viral DNA.
  • the nucleotide specificity of a domain comes from the two amino acids at positions 12 and 13 where Asn-Asn, Asn-Ile, His- Asp and Asn-Gly recognize guanine, adenine, cytosine and thymine, respectively. That pattern allows one to program TALENs to target various nucleic acids.
  • the programmable nuclease can comprise any type of engineered enzyme.
  • the programmable nuclease can comprise CRISPR enzymes derived from naturally occurring bacteria or phage.
  • a programmable nuclease can be a Cas effector protein (also referred to, interchangeably, as a Cas nuclease).
  • a guide nucleic acid (e.g., a crRNA) and Cas effector protein can form a CRISPR enzyme.
  • the programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid.
  • the programmable nuclease can comprise one or more amino acid modifications.
  • the programmable nuclease can be a nuclease derived from a CRISPR-Cas system.
  • the programmable nuclease can be a nuclease derived from recombineering.
  • the programmable nuclease further comprises a Cas enzyme.
  • the Cas enzyme is selected from the group consisting of Casl2, Casl3, Casl4, Casl4a, Casl4al, and CasPhi.
  • the programmable nuclease is Casl3.
  • the Casl3 is Casl3a, Casl3b, Casl3c, Casl3d, Casl3e, or Casl3f.
  • the programmable nuclease is Mad7 or Mad2.
  • the programmable nuclease is Casl2.
  • the Casl2 is Casl2a, Cas 12b, Cas 12c, Cas 12d, Casl2e, Casl2f, Cas 12g, Casl2h, Casl2i, Casl2j, or Cas 12k.
  • the programmable nuclease is Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ.
  • the Csml is also called smCmsl, miCmsl, obCmsl, or suCmsl.
  • Casl3a is also called C2c2.
  • CasZ is also called Casl4a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, or Casl4h.
  • the programmable nuclease is a type V CRISPR-Cas system.
  • the programmable nuclease is a type VI CRISPR-Cas system. Sometimes the programmable nuclease is a type III CRISPR-Cas system. In some cases, the programmable nuclease is from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rea), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp.
  • Leptotrichia shahii Lsh
  • Listeria seeligeri Lse
  • Psm Capnocytophaga canimorsus
  • Ca Lachnospiraceae bacterium
  • Bzo Bergeyella zoohelcum
  • Prevotella intermedia Pin
  • Prevotella buccae Pbu
  • Alistipes sp. Asp
  • Riemerella anatipestifer Ran
  • Prevotella aurantiaca Pau
  • Prevotella saccharolytica Psa
  • Pin2 Capnocytophaga canimorsus
  • Porphyromonas gulae Pgu
  • Prevotella sp Prevotella sp.
  • the Casl3 is at least one of LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, or LshCasl3a.
  • programmable nucleases comprise a Type V CRISPR/Cas protein.
  • Type V CRISPR/Cas proteins comprise nucleic acid cleavage activity.
  • Type V CRISPR/Cas proteins cleave or nick singlestranded nucleic acids, double, stranded nucleic acids, or a combination thereof.
  • Type V CRISPR/Cas proteins cleave single-stranded nucleic acids.
  • Type V CRISPR/Cas proteins cleave double-stranded nucleic acids.
  • Type V CRISPR/Cas proteins nick double-stranded nucleic acids.
  • guide nucleic acids of Type V CRISPR/Cas proteins hybridize to ssDNA or dsDNA.
  • the trans cleavage activity of Type V CRISPR/Cas protein is typically directed towards ssDNA.
  • the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain.
  • a catalytically inactive domain of a Type V CRISPR/Cas protein may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a wild-type nuclease domain of the Type V CRISPR/Cas protein. Said mutations may be present within a cleaving or active site of the nuclease.
  • the Type V Cas protein is a Cas protein.
  • a Cas protein can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid.
  • a programmable Cas nuclease may have a single active site in a RuvC domain that is capable of catalyzing pre-crRNA processing and nicking or cleaving of nucleic acids. This compact catalytic site may render the programmable Cas nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.
  • the programmable nuclease is a Type VI Cas protein.
  • the Type VI Cas protein is a programmable Cas 13 nuclease.
  • the general architecture of a Cas 13 protein includes an N-terminal domain and two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains.
  • the HEPN domains each comprise aR-X4-H motif. Shared features across Casl3 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase.
  • programmable Casl3 nucleases also consistent with the present disclosure include Casl3 nucleases comprising mutations in the HEPN domain that enhance the Cast 3 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains.
  • Programmable Casl3 nucleases consistent with the present disclosure also Cast 3 nucleases comprising catalytic components.
  • the Cas effector is a Cas 13 effector.
  • the Cas 13 effector is a Cas 13 a, a Cas 13b, a Cas 13c, a Cas 13d, or a Cas 13e effector protein.
  • the programmable nuclease comprises a Casl2 protein, wherein the Cas 12 enzyme binds and cleaves double stranded DNA and single stranded DNA.
  • programmable nuclease comprises a Casl3 protein, wherein the Casl3 enzyme binds and cleaves single stranded RNA.
  • programmable nuclease comprises a Casl4 protein, wherein the Casl4 enzyme binds and cleaves both double stranded DNA and single stranded DNA.
  • Table 1 provides illustrative amino acid sequences of programmable nucleases having trans-cleavage activity.
  • the programmable nuclease may comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-61 or 81-92.
  • the programmable nuclease may consist of an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one or SEQ ID Nos: 1-61 or 81-92.
  • the programmable nuclease may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any one of SEQ ID Nos: 1-61 or 81-92.
  • PCT/US2021/033271 PCT/US2021/035031
  • PCT/US2022/028865 all of which are herein incorporated by reference in their entirety.
  • the effector proteins comprise a RuvC domain (e.g., a partial RuvC domain).
  • the RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the protein.
  • An effector protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity.
  • an effector protein may include three partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds.
  • effector proteins comprise a recognition domain with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex.
  • the effector protein does not comprise a zinc finger domain.
  • the effector protein does not comprise an HNH domain.
  • Effector proteins disclosed herein may function as an endonuclease that catalyzes cleavage at a specific position (e.g., at a specific nucleotide within a nucleic acid sequence) in a target nucleic acid.
  • the target nucleic acid may be single stranded RNA (ssRNA), double stranded DNA (dsDNA) or single-stranded DNA (ssDNA).
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • ssDNA single-stranded DNA
  • the target nucleic acid is single-stranded DNA.
  • the target nucleic acid is single-stranded RNA.
  • the effector proteins may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof.
  • Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide nucleic acid (e.g., a dual gRNA or a sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guide nucleic acid.
  • Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide nucleic acid.
  • Trans cleavage activity is triggered by the hybridization of guide nucleic acid to the target nucleic acid.
  • nickase activity is a selective cleavage of one strand of a dsDNA.
  • Effector proteins of the present disclosure, dimers thereof, and multimeric complexes thereof may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid.
  • PAM protospacer adjacent motif
  • cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides of a 5’ or 3’ terminus of a PAM sequence.
  • a target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.
  • the Type V CRISPR/Cas protein has been modified (also referred to as an engineered protein).
  • a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS).
  • Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell.
  • the Type V CRISPR/Cas protein is codon optimized for a human cell.
  • Cas proteins are programmable nucleases used in the methods and systems disclosed herein.
  • Cas proteins can include any of the known Classes and Types of CRISPR/Cas enzymes.
  • Programmable nucleases disclosed herein include Class 1 Cas proteins, such as the Type I, Type IV, or Type III Cas proteins.
  • Programmable nucleases disclosed herein also include the Class 2 Cas proteins, such as the Type II, Type V, and Type VI Cas proteins.
  • Programmable nucleases included in the devices disclosed herein and methods of use thereof include a Type V or Type VI Cas proteins.
  • the programmable nuclease is a Type V Cas protein.
  • a Type V Cas effector protein comprises a RuvC domain, but lacks an HNH domain.
  • the RuvC domain of the Type V Cas effector protein comprises three patrial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains).
  • the three RuvC subdomains are located within the C-terminal half of the Type V Cas effector protein.
  • none of the RuvC subdomains are located at the N terminus of the protein.
  • the RuvC subdomains are contiguous.
  • the RuvC subdomains are not contiguous with respect to the primary amino acid sequence of the Type V Cas protein, but form a ruvC domain once the protein is produced and folds. In some instances, there are zero to about 50 amino acids between the first and second RuvC subdomains. In some instances, there are zero to about 50 amino acids between the second and third RuvC subdomains.
  • the Cas effector is a Casl4 effector.
  • the Casl4 effector is a Casl4a, Casl4al, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, or Casl4u effector.
  • the Cas effector is a CasPhi effector.
  • the Cas effector is a Casl2 effector.
  • the Casl2 effector is a Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, or Casl2j effector.
  • the Type V CRISPR/Cas protein comprises a Casl4 protein.
  • Casl4 proteins may comprise a bilobed structure with distinct amino-terminal and carboxy-terminal domains.
  • the amino- and carboxy-terminal domains may be connected by a flexible linker.
  • the flexible linker may affect the relative conformations of the amino- and carboxyl-terminal domains.
  • the flexible linker may be short, for example less than 10 amino acids, less than 8 amino acids, less than 6 amino acids, less than 5 amino acids, or less than 4 amino acids in length.
  • the flexible linker may be sufficiently long to enable different conformations of the amino- and carboxy -terminal domains among two Cast 4 proteins of a Cast 4 dimer complex (e.g., the relative orientations of the amino- and carboxy-terminal domains differ between two Casl4 proteins of a Casl4 homodimer complex).
  • the linker domain may comprise a mutation which affects the relative conformations of the amino- and carboxyl-terminal domains.
  • the linker may comprise a mutation which affects Casl4 dimerization. For example, a linker mutation may enhance the stability of a Cast 4 dimer.
  • the amino-terminal domain of a Casl4 protein comprises a wedge domain, a recognition domain, a zinc finger domain, or any combination thereof.
  • the wedge domain may comprise a multi-strand P-barrel structure.
  • a multi-strand P-barrel structure may comprise an oligonucleotide/oligosaccharide-binding fold that is structurally comparable to those of some Cast 2 proteins.
  • the recognition domain and the zinc finger domain may each (individually or collectively) be inserted between P-barrel strands of the wedge domain.
  • the recognition domain may comprise a 4-a-helix structure, structurally comparable but shorter than those found in some Casl2 proteins.
  • the recognition domain may comprise a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex.
  • a REC lobe may comprise a binding affinity for a PAM sequence in the target nucleic acid.
  • the amino-terminal may comprise a wedge domain, a recognition domain, and a zinc finger domain.
  • the carboxy-terminal may comprise a RuvC domain, a zinc finger domain, or any combination thereof.
  • the carboxy-terminal may comprise one RuvC and one zinc finger domain.
  • Casl4 proteins may comprise a RuvC domain or a partial RuvC domain.
  • the RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the Cast 4 protein.
  • a partial RuvC domain does not have any substrate binding activity or catalytic activity on its own.
  • a Casl4 protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity.
  • a Casl4 may include 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Casl4 protein, but form a RuvC domain once the protein is produced and folds.
  • a Casl4 protein may comprise a linker loop connecting a carboxy terminal domain of the Cast 4 protein with the amino terminal domain of the Cas 14 protein, and wherein the carboxy terminal domain comprises one or more RuvC domains and the amino terminal domain comprises a recognition domain.
  • Casl4 proteins may comprise a zinc finger domain.
  • a carboxy terminal domain of a Casl4 protein comprises a zinc finger domain.
  • an amino terminal domain of a Casl4 protein comprises a zinc finger domain.
  • the amino terminal domain comprises a wedge domain (e.g., a multi-P-barrel wedge structure), a zinc finger domain, or any combination thereof.
  • the carboxy terminal domain comprises the RuvC domains and a zinc finger domain, and the amino terminal domain comprises a recognition domain, a wedge domain, and a zinc finger domain.
  • Cast 4 proteins may be relatively small compared to many other Cas proteins, making them suitable for nucleic acid detection or gene editing. For instance, a Cas 14 protein may be less likely to adsorb to a surface or another biological species due to its small size. The smaller nature of these proteins also allows for them to be more easily packaged as a reagent in a system or assay, and delivered with higher efficiency as compared to other larger Cas proteins.
  • a Casl4 protein is 400 to 800 amino acid residues long, 400 to 600 amino acid residues long, 440 to 580 amino acid residues long, 460 to 560 amino acid residues long, 460 to 540 amino acid residues long, 460 to 500 amino acid residues long, 400 to 500 amino acid residues long, or 500 to 600 amino acid residues long. In some cases, a Casl4 protein is less than about 550 amino acid residues long. In some cases, a Casl4 protein is less than about 500 amino acid residues long.
  • a Cas 14 protein may function as an endonuclease that catalyzes cleavage at a specific position within a target nucleic acid.
  • a Casl4 protein is capable of catalyzing non-sequence-specific cleavage of a single stranded nucleic acid.
  • a Casl4 protein is activated to perform trans cleavage activity after binding of a guide nucleic acid with a target nucleic acid. This trans cleavage activity is also referred to as “collateral” or “transcollateral” cleavage. Trans cleavage activity may be non-specific cleavage of nearby single-stranded nucleic acid by the activated programmable nuclease, such as trans cleavage of reporters with a detection moiety.
  • the Type V CRISPR/Cas enzyme is a programmable Casl2 nuclease.
  • Type V CRISPR/Cas enzymes e.g., Casl2 or Casl4
  • a Casl2 nuclease of the present disclosure cleaves a nucleic acid via a single catalytic RuvC domain.
  • the RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Casl2 nucleases further comprise a recognition, or “REC” lobe.
  • a programmable Cast 2 nuclease can be a Cast 2a protein, a Cast 2b protein, Cast 2c protein, Cast 2d protein, or a Casl2e protein.
  • the programmable nuclease can be Cast 3. Sometimes the Cast 3 can be Cast 3 a, Cast 3b, Cast 3 c, Cast 3d, or Casl3e. In some cases, the programmable nuclease can be Mad7 or Mad2. In some cases, the programmable nuclease can be Casl2. Sometimes the Casl2 can be Casl2a, Casl2b, Casl2c, Casl2d, or Casl2e. In some cases, the Casl2 can be Casl2M08, which is a specific protein variant within the Casl2 protein family/classification).
  • the programmable nuclease can be Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ.
  • the Csml can also be also called smCmsl, miCmsl, obCmsl, or suCmsl.
  • Casl3a can also be also called C2c2.
  • CasZ can also be called Casl4a, Cast 4b, Cast 4c, Casl4d, Casl4e, Casl4f, Cast 4g, or Casl4h.
  • the programmable nuclease can be a type V CRISPR-Cas system.
  • the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system. Sometimes the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system.
  • the programmable nuclease can be from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rea), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp.
  • Psm Capnocytophaga canimorsus
  • Ca Lachnospiraceae bacterium
  • Bzo Bergeyella zoohelcum
  • Prevotella intermedia Pin
  • Prevotella buccae Pbu
  • Alistipes sp. Asp
  • Riemerella anatipestifer Ran
  • Prevotella aurantiaca Pau
  • Prevotella saccharolytica Psa
  • Pin2 Capnocytophaga canimorsus
  • Porphyromonas gulae Pgu
  • Prevotella sp Prevotella sp.
  • the Casl3 is at least one of LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, or LshCasl3a.
  • the trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid.
  • the trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target nucleic acid.
  • the target nucleic acid can be RNA or DNA.
  • a programmable nuclease as disclosed herein is an RNA-activated programmable RNA nuclease.
  • a programmable nuclease as disclosed herein is a DNA-activated programmable RNA nuclease.
  • a programmable nuclease is capable of being activated by a target RNA to initiate trans cleavage of an RNA reporter and is capable of being activated by a target DNA to initiate trans cleavage of an RNA reporter, such as a Type VI CRISPR/Cas enzyme (e.g., a Casl3 nuclease).
  • Casl3a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cast 3a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Cast 3a for trans cleavage of an RNA reporter.
  • An RNA reporter can be an RNA-based reporter.
  • the Cast 3a recognizes and detects ssDNA to initiate transcleavage of RNA reporters.
  • Multiple Casl3a isolates can recognize, be activated by, and detect target DNA, including ssDNA, upon hybridization of a guide nucleic acid with the target DNA.
  • Lbu-Casl3a and Lwa-Casl3a can both be activated to transcollaterally cleave RNA reporters by target DNA.
  • Type VI CRISPR/Cas enzyme e.g., a Casl3 nuclease, such as Casl3a
  • Casl3 nuclease such as Casl3a
  • DNA-activated programmable RNA nuclease detection of ssDNA can be robust at multiple pH values.
  • target ssDNA detection by Casl3 can exhibit consistent cleavage across a wide range of pH conditions, such as from a pH of 6.8 to a pH of 8.2.
  • target RNA detection by Casl3 can exhibit high cleavage activity of pH values from 7.9 to 8.2.
  • a DNA-activated programmable RNA nuclease that also is capable of being an RNA-activated programmable RNA nuclease can have DNA targeting preferences that are distinct from its RNA targeting preferences.
  • the optimal ssDNA targets for Cast 3a have different properties than optimal RNA targets for Cast 3 a.
  • gRNA performance on ssDNA can not necessarily correlate with the performance of the same gRNAs on RNA.
  • gRNAs can perform at a high level regardless of target nucleotide identity at a 3’ position on a target RNA sequence.
  • gRNAs can perform at a high level in the absence of a G at a 3’ position on a target ssDNA sequence.
  • target DNA detected by Casl3 disclosed herein can be directly taken from organisms or can be indirectly generated by nucleic acid amplification methods, such as PCR and LAMP or any amplification method described herein.
  • a target DNA such as a target ssDNA
  • a DNA-activated programmable RNA nuclease such as Cast 3 a
  • Key steps for the sensitive detection of a target DNA, such as a target ssDNA, by a DNA-activated programmable RNA nuclease, such as Cast 3 a can include: (1) production or isolation of DNA to concentrations above about 0.1 nM per reaction for in vitro diagnostics, (2) selection of a target sequence with the appropriate sequence features to enable DNA detection as these features are distinct from those required for RNA detection, and (3) buffer composition that enhances DNA detection.
  • the detection of a target DNA by a DNA-activated programmable RNA nuclease can be connected to a variety of readouts including fluorescence, lateral flow, electrochemistry, or any other readouts described herein.
  • Multiplexing of programmable DNA nuclease, such as a Type V CRISPR-Cas protein, with a DNA-activated programmable RNA nuclease, such as a Type VI protein, with a DNA reporter and an RNA reporter can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively.
  • Multiplexing of different RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing.
  • Methods for the generation of ssDNA for DNA- activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion.
  • DNA-activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein.
  • target ssDNA detection by Casl3a can be employed in a detection device as disclosed herein.
  • a programmable nuclease is referred to as an effector protein.
  • an effector protein disclosed herein is an engineered protein.
  • the engineered protein is not identical to a naturally-occurring protein.
  • the engineered protein may provide enhanced nuclease or nickase activity as compared to a naturally occurring nuclease or nickase.
  • some engineered proteins exhibit optimal activity at lower salinity and viscosity than the protoplasm of their bacterial cell of origin.
  • bacteria often comprise protoplasmic salt concentrations greater than 250 mM and room temperature intracellular viscosities above 2 centipoise
  • engineered proteins exhibit optimal activity (e.g., cis-cleavage activity) at salt concentrations below 150 mM and viscosities below 1.5 centipoise.
  • the present disclosure leverages these dependencies by providing engineered proteins in solutions optimized for their activity and stability.
  • compositions and systems described herein may comprise an engineered protein in a solution comprising a room temperature viscosity of less than about 15 centipoise, less than about 12 centipoise, less than about 10 centipoise, less than about 8 centipoise, less than about 6 centipoise, less than about 5 centipoise, less than about 4 centipoise, less than about 3 centipoise, less than about 2 centipoise, or less than about 1.5 centipoise.
  • compositions and systems may comprise an engineered protein in a solution comprising an ionic strength of less than about 500 mM, less than about 400 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 150 mM, less than about 100 mM, less than about 80 mM, less than about 60 mM, or less than about 50 mM.
  • Compositions and systems may comprise an engineered protein and an assay excipient, which may stabilize a reagent or product, prevent aggregation or precipitation, or enhance or stabilize a detectable signal (e.g., a fluorescent signal).
  • assay excipients include, but are not limited to, saccharides and saccharide derivatives (e.g., sodium carboxymethyl cellulose and cellulose acetate), detergents, glycols, polyols, esters, buffering agents, alginic acid, and organic solvents (e.g., DMSO).
  • An engineered protein may comprise a modified form of a wildtype counterpart protein.
  • the modified form of the wildtype counterpart may comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the programmable nuclease.
  • a nuclease domain e.g., RuvC domain
  • a Type V CRISPR/Cas protein may be deleted or mutated so that it is no longer functional or comprises reduced nuclease activity.
  • the modified form of the programmable nuclease may have less than 90 %, less than 80 %, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type counterpart.
  • Engineered proteins may have no substantial nucleic acid-cleaving activity.
  • Engineered proteins may be enzymatically inactive or “dead,” that is it may bind to a nucleic acid but not cleave it.
  • An enzymatically inactive protein may comprise an enzymatically inactive domain (e.g., inactive nuclease domain).
  • Enzymatically inactive may refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to the wild-type counterpart.
  • a dead protein may associate with an engineered guide nucleic acid to activate or repress transcription of a target nucleic acid sequence.
  • the enzymatically inactive protein is fused with a protein comprising recombinase activity.
  • a programmable nuclease is a fusion protein, wherein the fusion protein comprises a protein comprising the amino acid sequence of any one of SEQ ID NOs: 1- 61 or 81-92. In some instances, the fusion protein comprises a programmable nuclease and a fusion partner protein.
  • a fusion partner protein is also simply referred to herein as a fusion partner.
  • the fusion partner promotes the formation of a multimeric complex of the programmable nuclease.
  • the fusion partner is an additional programmable nuclease.
  • the multimeric complex comprising the programmable nuclease and the additional programmable nuclease binds a guide nucleic acid.
  • the programmable nucleases of the multimeric complex may bind the guide nucleic acid in an asymmetric fashion.
  • one programmable nuclease of the multimeric complex interacts more strongly with the guide nucleic acid than the additional programmable nuclease of the multimeric complex.
  • a programmable nuclease interacts more strongly with a target nucleic acid when it is complexed with the guide nucleic acid relative to when the programmable nuclease or the multimeric complex is not complexed with the guide nucleic acid.
  • the fusion partner has enzymatic activity in the presence of its enzyme substrate.
  • the fusion partner may comprise an enzyme such as horse radish peroxidase (HRP) which can catalyze a detectable color change reaction in the presence of its substrate (e.g., TMB).
  • HRP horse radish peroxidase
  • fusion partners include, but are not limited to, a protein that directly and/or indirectly provides for increased or decreased transcription and/or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.).
  • fusion partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.
  • a terminus of the programmable nuclease is linked to a terminus of the fusion partner through an amide bond.
  • a programmable nuclease is coupled to a fusion partner via a linker protein.
  • a programmable nuclease is coupled to a fusion partner via a linker protein.
  • the linker protein may have any of a variety of amino acid sequences.
  • a linker protein may comprise a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof.
  • the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility.
  • linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more portions that confer less flexible structure.
  • Suitable linkers include proteins of 4 linked amino acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length. These linkers may be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or may be encoded by a nucleic acid sequence encoding a fusion protein (e.g., an programmable nuclease coupled to a fusion partner).
  • linker proteins include glycine polymers (G)n (SEQ ID NO: 70), glycine-serine polymers (including, for example, (GS)n (SEQ ID NO: 71), GSGGSn (SEQ ID NO: 72), GGSGGSn (SEQ ID NO: 73), and GGGSn (SEQ ID NO: 74), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers.
  • Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 75), GGSGG (SEQ ID NO: 76), GSGSG (SEQ ID NO: 77), GSGGG (SEQ ID NO: 78), GGGSG (SEQ ID NO: 79), and GSSSG (SEQ ID NO: 80).
  • compositions and systems comprising at least one of an engineered Cas protein and an engineered guide nucleic acid, which may simply be referred to herein as a Cas protein and a guide nucleic acid, respectively.
  • an engineered Cas protein and an engineered guide nucleic acid refer to a Cas protein and a guide nucleic acid, respectively, that are not found in nature.
  • systems and compositions comprise at least one non-naturally occurring component.
  • compositions and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid.
  • compositions and systems comprise at least two components that do not naturally occur together.
  • compositions and systems may comprise a guide nucleic acid comprising a repeat region and a spacer region which do not naturally occur together.
  • compositions and systems may comprise a guide nucleic acid and a Cas protein that do not naturally occur together.
  • a Cas protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes Cas proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.
  • the guide nucleic acid may comprise a non-natural nucleobase sequence.
  • the non-natural sequence is a nucleobase sequence that is not found in nature.
  • the non-natural sequence may comprise a portion of a naturally occurring sequence, wherein the portion of the naturally occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence.
  • the guide nucleic acid may comprise two naturally occurring sequences arranged in an order or proximity that is not observed in nature.
  • compositions and systems comprise a ribonucleotide complex comprising a programmable nuclease and a guide nucleic acid that do not occur together in nature.
  • Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together.
  • an engineered guide nucleic acid may comprise a sequence of a naturally occurring repeat region and a spacer region that is complementary to a naturally occurring eukaryotic sequence.
  • the engineered guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism.
  • An engineered guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different.
  • the guide nucleic acid may comprise a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid.
  • an engineered guide nucleic acid may comprise a naturally occurring crRNA and tracrRNA coupled by a linker sequence.
  • compositions and systems described herein comprise an engineered Cas protein that is similar to a naturally occurring Cas protein.
  • the engineered Cas protein may lack a portion of the naturally occurring Cas protein.
  • the Cas protein may comprise a mutation relative to the naturally-occurring Cas protein, wherein the mutation is not found in nature.
  • the Cas protein may also comprise at least one additional amino acid relative to the naturally- occurring Cas protein.
  • the Cas protein may comprise an addition of a nuclear localization signal relative to the natural occurring Cas protein.
  • the nucleotide sequence encoding the Cas protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.
  • thermostable programmable nucleases a programmable nuclease is referred to as a programmable nuclease.
  • a programmable nuclease may be thermostable.
  • known programmable nucleases e.g., Casl2 nucleases
  • a thermostable protein may have enzymatic activity, stability, or folding comparable to those at 37 °C.
  • the trans cleavage activity (e.g., the maximum trans cleavage rate as measured by fluorescent signal generation) of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 50% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 55% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 60 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 65% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 70 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 75% of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 80% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 85% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 90% of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 95% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40°C may be at least 100% of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 1-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 2-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 3-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 4-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 5-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 6-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 7-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 8-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 9-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 10-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 40 °C may be at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 50 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 55 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 60 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 65 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 70 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 75 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 80 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 85 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 90 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 95 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 100 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 1-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 2-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 3-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 4-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 5-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 6-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 7-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 8-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45°C may be at least 9-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45 °C may be at least 10-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 45°C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 50 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 55 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 60 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 65 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 70 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 75 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 80 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 85 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 90 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 95 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 100 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 1-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 2-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 3-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 4-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 5-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 6-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 7-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 8-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 9-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 10-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 50 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 55 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 60 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 65 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 70 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 75 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 80 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 85 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 90 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 95 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 100 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 1-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 2-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 3-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 4-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 5-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 6-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 7-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 8-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 9-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 10-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 55 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 50 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 55 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 60 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 65 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 70 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 75 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 80 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 85 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 90 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 95 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 100 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 1-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 2-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 3-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 4-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 5-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 6-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 7-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 8-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 9-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 10-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 50 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 55 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 60 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 65 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 70 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 75 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 80 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 85 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 90 % of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 95 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 100 % of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 1-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 2-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 3-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 4-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 5-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 6-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 7-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 8-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 9-fold of that at 37 °C. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 10-fold of that at 37 °C.
  • the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • 80 °C, or more may be at least 50, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold , at least 3-fold , at least 4-fold , at least 5-fold , at least 6-fold , at least 7-fold , at least 8-fold , at least 9-fold , at least 10-fold , at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
  • the trans cleavage activity may be measured against a negative control in a trans cleavage assay.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 37 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 37 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 40 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 40 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 45 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 45 °C may be at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 50 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 55 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 55 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 60 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 65 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
  • the trans cleavage activity of a programmable nuclease against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 11-fold, at least 12- fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
  • the reporters described herein can be RNA reporters.
  • the RNA reporters can comprise at least one ribonucleic acid and a detectable moiety.
  • a programmable nuclease probe or a CRISPR probe comprising a programmable nuclease can recognize and detect ssDNA and, further, can specifically trans-cleave RNA reporters.
  • the detection of the target nucleic acid in the sample can indicate the presence of the disease (or disease-causing agent) in the sample and can provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual.
  • Cleavage of a reporter i.e., a protein-nucleic acid or detector nucleic acid
  • a reporter i.e., a protein-nucleic acid or detector nucleic acid
  • the signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample.
  • cleavage of the reporter can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal.
  • Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample.
  • the sensors and detectors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor or detector suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
  • optical sensors e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies
  • SPR surface plasmon resonance
  • interferometric sensors or any other type of sensor or detector suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
  • the present disclosure provides a method for target detection.
  • the method can comprise sample collection.
  • the method can further comprise sample preparation.
  • the method can further comprise detection of one or more target molecules in the collected and prepared sample.
  • sample preparation can include nucleic acid amplification and the target molecules can include target amplicons.
  • the present disclosure provides a detection device for target detection.
  • the detection device can be configured for multiplexed target detection.
  • the detection device can be used to collect one or more samples, prepare or process the one or more samples for detection, and optionally divide the one or more samples into a plurality of droplets, aliquots, volumes, or subsamples for amplification of one or more target sequences or target nucleic acids.
  • the target sequences may comprise, for example, a biological sequence.
  • the biological sequence can comprise a nucleic acid sequence (e.g., a DNA sequence or an RNA sequence).
  • the target sequences are associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest.
  • the detection device can be configured to amplify target nucleic acids contained within the plurality of aliquots or subsamples.
  • the detection device can be configured to amplify the target sequences or target nucleic acids contained within the plurality volumes by individually processing each of the plurality of volumes (e.g., by using a thermocycling process or any other suitable amplification process as described in greater detail below).
  • the plurality of volumes can undergo separate thermocycling processes.
  • the thermocycling processes can occur simultaneously.
  • the thermocycling processes can occur at different times for each droplet or volume.
  • amplification is an isothermal amplification process.
  • the detection device can be configured to provide the sample aliquots, volumes, or subsamples to a detection region of the device, or otherwise partition a sample into separate volumes within a plurality of partitions within the detection region (e.g., a plurality of chambers or wells).
  • plurality of partitions are fluidically connected, such as by channels and/or valves (e.g., capillary valves).
  • the detection region may comprise a plurality of programmable nuclease probes.
  • the detection chamber can be configured to direct the aliquots, volumes, or subsamples along one or more fluid flow paths such that the aliquots, volumes, or subsamples are in contact with the one or more programmable nuclease probes.
  • the instrument and/or detection device can comprise one or more sensors or detectors.
  • the one or more sensors or detectors of the instrument and/or detection device can be configured to detect one or more signals that are generated after one or more programmable nucleases of the one or more programmable nuclease probes become activated due to a binding of a guide nucleic acid of the programmable nuclease probes with a target nucleic acid present in the sample or amplicon thereof.
  • the activated programmable nuclease can bind or cleave the target nucleic acid, which can result in a trans cleavage activity.
  • Trans cleavage activity can be a non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of reporter nucleic acids with a detection moiety.
  • the detection moiety can be released or separated from the reporter, thereby generating one or more detectable signals.
  • the one or more sensors or detectors of the instrument or detection device can be configured to register and/or process the one or more detectable signals to confirm a presence and/or an absence of a particular target (e.g., a target nucleic acid) in a sample.
  • the one or more programmable nuclease probes of the detection device can be configured for multiplexed detection.
  • each programmable nuclease probe can be configured to detect a particular target.
  • each programmable nuclease probe can be configured to detect a plurality of targets.
  • a first programmable nuclease probe can be configured to detect a first target or a first set of targets, and a second programmable nuclease probe can be configured to detect a second target or a second set of targets.
  • a first programmable nuclease probe can be configured to detect a first set of targets, and a second programmable nuclease probe can be configured to detect a second set of targets.
  • the programmable nuclease probes of the present disclosure can be used to detect a plurality of different target sequences or target nucleic acids.
  • the sample provided to the detection device can comprise a plurality of target sequences or target nucleic acids.
  • the sample provided to the detection device can comprise multiple classes of target sequences or target nucleic acids.
  • Each class of target sequences or class of target nucleic acids can comprise a plurality of target sequences or target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample.
  • each programmable nuclease probe can be used to detect a particular class of target sequences, or a particular class of target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample.
  • two or more programmable nuclease probes can be used to detect different classes of target sequences or different classes of target nucleic acids. In such cases, the two or more programmable nuclease probes can comprise different sets or classes of guide nucleic acids complexed to the programmable nucleases of the probes.
  • Guide nucleic acids are compatible for use in the devices described herein and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, and reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions).
  • the guide nucleic acid binds to the single stranded or double stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein.
  • the guide nucleic acid can bind to the single stranded or double stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment.
  • SNP single nucleotide polymorphism
  • the guide nucleic acid binds to the single stranded or double stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein.
  • the guide nucleic acid is complementary to the target nucleic acid or a portion thereof.
  • the guide nucleic acid binds specifically to the target nucleic acid.
  • the target nucleic acid may be a RNA, DNA, or synthetic nucleic acids.
  • a guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid.
  • a guide nucleic acid can be a crRNA.
  • a guide nucleic acid may comprise a crRNA and tracrRNA.
  • the guide nucleic acid can bind specifically to the target nucleic acid.
  • the guide nucleic acid is not naturally occurring.
  • the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence.
  • the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids.
  • the target nucleic acid can be designed and made to provide desired functions.
  • the targeting region of a guide nucleic acid is 20 nucleotides in length.
  • the targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the targeting region ofthe guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to
  • the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt.
  • the guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid.
  • the guide nucleic acid in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid.
  • the guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.
  • the guide nucleic acid in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.
  • the guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest.
  • the guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18, for example.
  • guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay.
  • the pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein.
  • the pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein.
  • the tiling for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential.
  • a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some reporters of a population of reporters. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that may be caused by multiple organisms.
  • Reporters which can be referred to interchangeably reporter molecules, or detector nucleic acids, described herein are compatible for use in the devices described herein and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions).
  • compositions disclosed herein e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof
  • a reporter comprising a single stranded nucleic acid and a detection moiety, wherein the reporter is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal.
  • a detector nucleic acid is used interchangeably with reporter or reporter molecule.
  • the reporter comprises a single-stranded nucleic acid.
  • the reporter comprises a double-stranded nucleic acid.
  • the reporter can comprise a single-stranded nucleic acid coupled to a double-stranded nucleic acid.
  • the reporter comprises a singlestranded nucleic acid comprising deoxyribonucleotides.
  • the reporter comprises a double-stranded nucleic acid comprising deoxyribonucleotides. In some cases, the reporter comprises a single-stranded nucleic acid comprising ribonucleotides.
  • the reporter can comprise a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the reporter comprises a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the reporter may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position.
  • the reporter may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the reporter may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the reporter has only ribonucleotide residues.
  • the reporter has only deoxyribonucleotide residues.
  • the reporter may comprise nucleotides resistant to cleavage by the programmable nuclease described herein.
  • the reporter may comprise synthetic nucleotides.
  • the reporter may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue.
  • the reporter is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length.
  • the reporter is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length.
  • the reporter may comprise at least one uracil ribonucleotide. In some cases, the reporter may comprise at least two uracil ribonucleotides.
  • the reporter has only uracil ribonucleotides.
  • the reporter may comprise at least one adenine ribonucleotide.
  • the reporter may comprise at least two adenine ribonucleotide.
  • the reporter has only adenine ribonucleotides.
  • the reporter may comprise at least one cytosine ribonucleotide.
  • the reporter may comprise at least two cytosine ribonucleotide.
  • the reporter may comprise at least one guanine ribonucleotide.
  • the reporter may comprise at least two guanine ribonucleotide.
  • a reporter can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof.
  • the reporter is from 5 to 12 nucleotides in length.
  • the reporter is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the reporter is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • a reporter can be 5, 8, or 10 nucleotides in length.
  • a reporter can be 10 nucleotides in length.
  • the reporter may comprise a nucleic acid and a detection moiety.
  • a reporter is connected to a surface by a linkage.
  • a reporter may comprise at least one of a nucleic acid, a chemical functionality, a detection moiety, a quenching moiety, or a combination thereof.
  • a reporter is configured for the detection moiety to remain immobilized to the surface and the quenching moiety to be released into solution upon cleavage of the reporter.
  • a reporter is configured for the quenching moiety to remain immobilized to the surface and for the detection moiety to be released into solution, upon cleavage of the reporter.
  • the detection moiety is at least one of a label, a polypeptide, a dendrimer, or a nucleic acid or a combination thereof.
  • the reporter contains a label.
  • label may be FITC, DIG, TAMRA, Cy5, AF594, or Cy3.
  • the label may comprise a dye, a nanoparticle configured to produce a signal, or the like.
  • the dye may be a fluorescent dye.
  • the at least one chemical functionality may comprise biotin. In some embodiments, the at least one chemical functionality may be configured to be captured by a capture probe.
  • the at least one chemical functionality may comprise biotin and the capture probe may comprise anti-biotin, streptavidin, avidin or other molecule configured to bind with biotin.
  • the dye is the chemical functionality.
  • a capture probe may comprise a molecule that is complementary to the chemical functionality of the reporter.
  • the capture antibodies are anti-FITC, anti-DIG, anti-TAMRA, anti-Cy5, anti-AF594, or any other appropriate capture antibody capable of binding the detection moiety or conjugate.
  • the detection moiety can be the chemical functionality.
  • the reporter may comprise a quenching moiety.
  • a quenching moiety is any entity that decreases the fluorescence intensity of a given substance.
  • Exemplary embodiments of reporters, labels, quenchers, chemical functionalities, detection moieties, dendrimers, quenching moieties and other reporter elements are described in: PCT/US2021/033271; PCT/US2021/035031, and PCT/US2022/028865, all of which are herein incorporated by reference in their entirety.
  • the reporter comprises a detection moiety and a quenching moiety.
  • the reporter comprises a cleavage site, wherein the detection moiety is located at a first site on the reporter and the quenching moiety is located at a second site on the reporter, wherein the first site and the second site are separated by the cleavage site.
  • the quenching moiety is a fluorescence quenching moiety.
  • the quenching moiety is 5' to the cleavage site and the detection moiety is 3' to the cleavage site.
  • the detection moiety is 5' to the cleavage site and the quenching moiety is 3' to the cleavage site.
  • the quenching moiety is at the 5' terminus of the nucleic acid of a reporter.
  • the detection moiety is at the 3' terminus of the nucleic acid of a reporter. In some cases, the detection moiety is at the 5' terminus of the nucleic acid of a reporter. In some cases, the quenching moiety is at the 3' terminus of the nucleic acid of a reporter.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP,
  • Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), betagalactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, CE ⁇ - glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • GAL betagalactosidase
  • glucose-6-phosphate dehydrogenase beta-N-acetylglucosaminidase
  • CE ⁇ - glucuronidase invertase
  • Xanthine Oxidase firefly luciferase
  • glucose oxidase GO
  • the detection moiety comprises an invertase.
  • the substrate of the invertase may be sucrose.
  • a DNS reagent may be included in the system to produce a colorimetric change when the invertase converts sucrose to glucose.
  • the reporter nucleic acid and invertase are conjugated using a heterobifunctional linker via sulfo-SMCC chemistry.
  • Suitable fluorophores may provide a detectable fluorescence signal in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).
  • fluorophores are fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester).
  • the fluorophore may be an infrared fluorophore.
  • the fluorophore may emit fluorescence in the range of 500 nm and 720 nm.
  • the fluorophore emits fluorescence at a wavelength of 700 nm or higher. In other cases, the fluorophore emits fluorescence at about 665 nm. In some cases, the fluorophore emits fluorescence in the range of 500 nm to 520 nm, 500 nm to 540 nm, 500 nm to 590 nm, 590 nm to 600 nm, 600 nm to 610 nm,
  • the fluorophore emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm.
  • Systems may comprise a quenching moiety.
  • a quenching moiety may be chosen based on its ability to quench the detection moiety.
  • a quenching moiety may be a non-fluorescent fluorescence quencher.
  • a quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 nm and 720 nm.
  • a quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. In some cases, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher.
  • the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some cases, the quenching moiety quenches a detection moiety that emits fluorescence in the range of 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 690 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm.
  • the quenching moiety quenches a detection moiety that emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm.
  • a quenching moiety may quench fluorescein amidite, 6- Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester).
  • a quenching moiety may be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher.
  • a quenching moiety may quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).
  • a quenching moiety may be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of the quenching moieties described herein may be from any commercially available source, may be an alternative with a similar function, a generic, or a non-trade name of the quenching moieties listed.
  • the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some cases, the detection moiety comprises an infrared (IR) dye. In some cases, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a protein. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some instances, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.
  • FRET fluorescence resonance energy transfer
  • a detection moiety may be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
  • a nucleic acid of a reporter sometimes, is protein-nucleic acid that is capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid.
  • a calorimetric signal is heat produced after cleavage of the nucleic acids of a reporter.
  • a calorimetric signal is heat absorbed after cleavage of the nucleic acids of a reporter.
  • a potentiometric signal for example, is electrical potential produced after cleavage of the nucleic acids of a reporter.
  • An amperometric signal may be movement of electrons produced after the cleavage of nucleic acid of a reporter.
  • the signal is an optical signal, such as a colorimetric signal or a fluorescence signal.
  • An optical signal is, for example, a light output produced after the cleavage of the nucleic acids of a reporter.
  • an optical signal is a change in light absorbance between before and after the cleavage of nucleic acids of a reporter.
  • a piezo-electric signal is a change in mass between before and after the cleavage of the nucleic acid of a reporter.
  • Other methods of detection can also be used, such as optical imaging, surface plasmon resonance (SPR), and/or interferometric sensing.
  • the detectable signal may be a colorimetric signal or a signal visible by eye.
  • the detectable signal may be fluorescent, electrical, chemical, electrochemical, or magnetic.
  • a detectable signal (e.g., a first detectable signal) may be generated by binding of the detection moiety to the capture molecule in the detection region, where the detectable signal indicates that the sample contained the target nucleic acid.
  • systems are capable of detecting more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter nucleic acid.
  • the detectable signal may be generated directly by the cleavage event.
  • the detectable signal may be generated indirectly by the cleavage event.
  • the detectable signal is not a fluorescent signal.
  • the detectable signal may be a colorimetric or color-based signal.
  • the detected target nucleic acid may be identified based on its spatial location on the detection region of the support medium.
  • a second detectable signal may be generated in a spatially distinct location than a first detectable signal when two or more detectable signals are generated.
  • the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes.
  • real-time detection of the Cas reaction can be achieved using fluorescence, electrochemical detection, and/or electrochemiluminescence.
  • the detectable signals can be detected and analyzed in various ways.
  • the detectable signals can be detected using an imaging device.
  • the imaging device can a digital camera, such a digital camera on a mobile device.
  • the mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement device can be used to detect and/or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein.
  • the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope).
  • a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope).
  • the reporter is an enzyme-nucleic acid.
  • the enzyme may be sterically hindered when present as in the enzyme-nucleic acid, but then functional upon cleavage from the nucleic acid by the programmable nuclease.
  • the enzyme is an enzyme that produces a reaction with an enzyme substrate.
  • An enzyme can be invertase.
  • the substrate of invertase is sucrose and DNS reagent.
  • the reporter is a substrate-nucleic acid.
  • the substrate is a substrate that produces a reaction with an enzyme. Release of the substrate upon cleavage by the programmable nuclease may free the substrate to react with the enzyme.
  • a reporter may be attached to a solid support.
  • the solid support for example, is a surface.
  • a surface can be an electrode.
  • the solid support is a bead.
  • the bead is a magnetic bead.
  • the detection moiety is liberated from the solid support and interacts with other mixtures.
  • the detection moiety is an enzyme, and upon cleavage of the nucleic acid of the enzyme-nucleic acid, the enzyme flows through a chamber into a mixture comprising the substrate. When the enzyme meets the enzyme substrate, a reaction occurs, such as a colorimetric reaction, which is then detected.
  • the detection moiety is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.
  • the reporter comprises a nucleic acid conjugated to an affinity molecule which is in turn conjugated to the fluorophore (e.g., nucleic acid - affinity molecule - fluorophore) or the nucleic acid conjugated to the fluorophore which is in turn conjugated to the affinity molecule (e.g., nucleic acid - fluorophore - affinity molecule).
  • a linker conjugates the nucleic acid to the affinity molecule.
  • a linker conjugates the affinity molecule to the fluorophore.
  • a linker conjugates the nucleic acid to the fluorophore.
  • a linker can be any suitable linker known in the art.
  • the nucleic acid of the reporter can be directly conjugated to the affinity molecule and the affinity molecule can be directly conjugated to the fluorophore or the nucleic acid can be directly conjugated to the fluorophore and the fluorophore can be directly conjugated to the affinity molecule.
  • “directly conjugated” indicates that no intervening molecules, polypeptides, proteins, or other moieties are present between the two moieties directly conjugated to each other.
  • a reporter comprises a nucleic acid directly conjugated to an affinity molecule and an affinity molecule directly conjugated to a fluorophore - no intervening moiety is present between the nucleic acid and the affinity molecule and no intervening moiety is present between the affinity molecule and the fluorophore.
  • the affinity molecule can be biotin, avidin, streptavidin, or any similar molecule.
  • the reporter comprises a substrate-nucleic acid.
  • the substrate may be sequestered from its cognate enzyme when present as in the substrate-nucleic acid, but then is released from the nucleic acid upon cleavage, wherein the released substrate can contact the cognate enzyme to produce a detectable signal.
  • the substrate is sucrose and the cognate enzyme is invertase, and a DNS reagent can be used to monitor invertase activity.
  • a reporter may be a hybrid nucleic acid reporter.
  • a hybrid nucleic acid reporter comprises a nucleic acid with at least one deoxyribonucleotide and at least one ribonucleotide.
  • the nucleic acid of the hybrid nucleic acid reporter can be of any length and can have any mixture of DNAs and RNAs. For example, in some cases, longer stretches of DNA can be interrupted by a few ribonucleotides. Alternatively, longer stretches of RNA can be interrupted by a few deoxyribonucleotides. Alternatively, every other base in the nucleic acid may alternate between ribonucleotides and deoxyribonucleotides.
  • hybrid nucleic acid reporter is increased stability as compared to a pure RNA nucleic acid reporter.
  • a hybrid nucleic acid reporter can be more stable in solution, lyophilized, or vitrified as compared to a pure DNA or pure RNA reporter.
  • the reporter can be lyophilized or vitrified.
  • the reporter can be suspended in solution or immobilized on a surface.
  • the reporter can be immobilized, dried, or otherwise deposited on the surface of a chamber in a device as disclosed herein.
  • the reporter is immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they can be held in position by a magnet placed below the chamber.
  • the reporter is a single-stranded nucleic acid comprising deoxyribonucleotides. In some cases, the reporter nucleic acid is a single-stranded nucleic acid sequence comprising ribonucleotides.
  • the nucleic acid of a reporter may be a single-stranded nucleic acid sequence comprising at least one ribonucleotide. In some cases, the nucleic acid of a reporter is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site.
  • the nucleic acid of a reporter comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ribonucleotide residues at an internal position.
  • the nucleic acid of a reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position.
  • the reporter may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position.
  • the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues.
  • the nucleic acid of a reporter has only ribonucleotide residues. In some cases, the nucleic acid of a reporter has only deoxyribonucleotide residues. In some cases, the nucleic acid comprises nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the nucleic acid of a reporter comprises synthetic nucleotides.
  • the nucleic acid of a reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue. [0168] In some cases, the nucleic acid of a reporter comprises at least one uracil ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two uracil ribonucleotides. Sometimes the nucleic acid of a reporter has only uracil ribonucleotides. In some cases, the nucleic acid of a reporter comprises at least one adenine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two adenine ribonucleotide.
  • the nucleic acid of a reporter has only adenine ribonucleotides. In some cases, the nucleic acid of a reporter comprises at least one cytosine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two cytosine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least one guanine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two guanine ribonucleotide. In some instances, a nucleic acid of a reporter comprises a single unmodified ribonucleotide. In some instances, a nucleic acid of a reporter comprises only unmodified ribonucleotides. In some instances, a nucleic acid of a reporter comprises only unmodified deoxyribonucleotides.
  • the nucleic acid of a reporter is 5 to 20, 5 to 15, 5 to 10, 7 to 20, 7 to 15, or 7 to 10 nucleotides in length. In some cases, the nucleic acid of a reporter is 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 13 to 20, 15 to 20, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8, nucleotides in length. In some cases, the nucleic acid of a reporter is 5 to 12 nucleotides in length.
  • the reporter nucleic acid is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides in length.
  • the reporter nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, at least 29, or at least 30 nucleotides in length.
  • a reporter For cleavage by a programmable nuclease comprising Casl3, a reporter can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Casl2, a reporter can be 10 nucleotides in length.
  • systems comprise a plurality of reporters.
  • the plurality of reporters may comprise a plurality of signals.
  • systems comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 reporters.
  • systems comprise a Type V CRISPR/Cas protein and a reporter nucleic acid configured to undergo transcollateral cleavage by the Type V CRISPR/Cas protein.
  • Transcollateral cleavage of the reporter may generate a signal from the reporter or alter a signal from the reporter.
  • the signal is an optical signal, such as a fluorescence signal or absorbance band.
  • Transcollateral cleavage of the reporter may alter the wavelength, intensity, or polarization of the optical signal.
  • the reporter may comprise a fluorophore and a quencher, such that transcollateral cleavage of the reporter separates the fluorophore and the quencher thereby increasing a fluorescence signal from the fluorophore.
  • detection of reporter cleavage to determine the presence of a target nucleic acid sequence may be referred to as 'DETECTR'.
  • a method of assaying for a target nucleic acid in a sample comprising contacting the target nucleic acid with a programmable nuclease, a non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter nucleic acid, and assaying for a change in a signal, wherein the change in the signal is produced by cleavage of the reporter nucleic acid.
  • a programmable nuclease e.g., a Type V CRISPR/Cas protein as disclosed herein
  • systems comprise an excess of reporter(s), such that when the system is operated and a solution of the system comprising the reporter is combined with a sample comprising a target nucleic acid, the concentration of the reporter in the combined solution-sample is greater than the concentration of the target nucleic acid.
  • the sample comprises amplified target nucleic acid.
  • the sample comprises an unamplified target nucleic acid.
  • the concentration of the reporter is greater than the concentration of target nucleic acids and non-target nucleic acids.
  • the nontarget nucleic acids may be from the original sample, either lysed or unlysed.
  • the non-target nucleic acids may comprise byproducts of amplification.
  • systems comprise a reporter wherein the concentration of the reporter in a solution 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold excess of total nucleic acids.
  • immobilized reporter systems e.g., for detection of a target nucleic acid or a plurality of target nucleic acids.
  • systems comprise a Type V CRISPR/Cas protein and a reporter configured to undergo transcollateral cleavage by the Type V CRISPR/Cas protein.
  • systems comprise a Type VI CRISPR/Cas protein and a reporter configured to undergo transcollateral cleavage by the Type VI CRISPR/Cas protein.
  • Transcollateral cleavage of the reporter may generate a signal from the reporter, alter a signal from the reporter, or trigger a downstream reaction capable of generating or changing a signal in response to cleavage of the reporter and release of a detection moiety therefrom.
  • the signal is an optical signal, such as a fluorescence signal or absorbance signal.
  • Transcollateral cleavage of the reporter may alter the wavelength, intensity, and/or polarization of the optical signal.
  • the reporter may comprise a fluorophore and a quencher, such that transcollateral cleavage of the reporter separates the fluorophore and the quencher thereby increasing a fluorescence signal from the fluorophore.
  • a method of assaying for a target nucleic acid in a sample comprising contacting the target nucleic acid with a programmable nuclease, a non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter, and assaying for a change in a signal, wherein the change in the signal is produced by cleavage of the reporter.
  • Reporter systems disclosed herein may comprise one or more reporters. Described herein are compositions and methods of use thereof comprising one or more reporter molecules.
  • the one or more reporter molecules comprise one or more different reporter molecules.
  • the one or more reporter molecules comprise a first reporter molecule, a second reporter molecule, a third reporter molecule, and/or more reporter molecules or a plurality of each reporter molecule wherein each reporter molecule can be present in multiple copies (e.g., at a predefined concentration) in the composition.
  • the compositions and methods comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • a reporter may comprise a single stranded nucleic acid and a detection moiety (e.g., a labeled single stranded RNA reporter), wherein the nucleic acid is capable of being cleaved by a programmable nuclease (e.g., a Type V or Type VI CRISPR/Cas protein as disclosed herein) or a multimeric complex thereof, releasing the detection moiety, and, generating a detectable signal.
  • a detection moiety e.g., a labeled single stranded RNA reporter
  • the reporter additionally comprises a double stranded nucleic acid.
  • reporter is used interchangeably with “reporter molecule”.
  • the programmable nucleases disclosed herein, activated upon hybridization of a guide RNA to a target nucleic acid, may cleave the reporter. Cleaving the “reporter” may be referred to herein as cleaving the “reporter nucleic acid,” the “reporter molecule,” or the “nucleic acid of the reporter.”
  • Reporters may comprise RNA.
  • Reporters may comprise DNA. Reporters may be double-stranded. Reporters may be singlestranded.
  • a reporter may be immobilized on a substrate.
  • a reporter may be immobilized to a surface of the substrate.
  • the reporter may be immobilized to a detection location of a substrate.
  • the reporter may be immobilized on the substrate.
  • the reporter can be attached to a solid support.
  • the solid support for example, is a surface.
  • a surface can be an electrode.
  • the solid support is a bead.
  • the bead is a magnetic bead.
  • the surface can also be an array or a slide.
  • the reporter comprising a nucleic acid in some cases, may be immobilized at the 5 ’end of the nucleic acid. In some cases, the reporter may be immobilized at the 3’ end of the nucleic acid. In some cases, the reporter may be immobilized at the 5’ and 3’ end of the nucleic acid.
  • a reporter may be immobilized to a substrate via covalent bonding.
  • the reporter may comprise a thiol or an amine group for immobilization.
  • the one or more detection reagents can be immobilized in discrete detection locations using NHS-amine chemistry as described herein.
  • a primary amine-modified guide nucleic acid and a primary amine-modified reporter may be conjugated to an NHS-coated surface of the detection region.
  • the amine may form an amide bond with the substrate.
  • the substrate may comprise graphene oxide.
  • NHS-amine in some cases, may have a structure of [0181]
  • the immobilization moiety of a reporter may comprise a thiol group. The thiol group may form an Au-S bond with the substrate.
  • the substrate may comprise gold.
  • the one or more detection reagents may be immobilized using maleimide- thiol chemistry as described herein.
  • a thiol-modified guide nucleic acid and a thiol- modified reporter may be conjugated to a maleimide-coated surface of the detection region.
  • Thiol group in some cases, may have a structure of S-H, wherein S is sulfur.
  • a reporter may be immobilized to a substrate via non-covalent bonding.
  • the one or more detection reagents may be immobilized using avidin/streptavidin-biotin chemistry as described herein.
  • a biotinylated reporter and a biotinylated guide nucleic acid may be immobilized to a streptavidin-coated surface of the detection region.
  • the immobilization of the reporter on a substrate may comprise an immobilization moiety.
  • the reporter may comprise an amino group moiety, a peptide moiety, a polypeptide moiety, or a protein moiety.
  • the amino group moiety, peptide moiety, polypeptide moiety, or protein moiety may be the immobilization moiety.
  • the immobilization moiety may comprise an amino modifier.
  • the immobilization moiety may be 5’ or 3’ of the nucleic acid of the reporter.
  • a reporter may be immobilized by surface adsorption.
  • the reporter may be immobilized on the surface via electrostatic interaction between the reporter and the surface.
  • a reporter may comprise a negative charge and a surface may comprise a positive charge.
  • the surface of a substrate may be coated with a material.
  • the coated material may comprise polyamine, poly-L-lysine, polypyrrole, polyaniline, polyethyleneimine, or a combination thereof.
  • the immobilization moiety may also be used to immobilize a guide nucleic acid.
  • the immobilization moiety can be located at an end of a guide nucleic acid.
  • the immobilization moiety can be located at the 5’ end of a guide nucleic acid.
  • the immobilization moiety can be located at the 3’ end of a guide nucleic acid.
  • the immobilization moiety can be located at the 5’ and 3’ end of a guide nucleic acid.
  • a reporter may comprise a nucleic acid.
  • the nucleic acid may have a polynucleotide sequence.
  • the polynucleotide sequence may comprise about 10 nucleotides.
  • the polynucleotide sequence may comprise about 11 nucleotides.
  • the polynucleotide sequence may comprise about 12 nucleotides.
  • the polynucleotide sequence may comprise about 13 nucleotides.
  • the polynucleotide sequence may comprise about 14 nucleotides.
  • the polynucleotide sequence may comprise about 15 nucleotides.
  • the polynucleotide sequence may comprise about 16 nucleotides. In some cases, the polynucleotide sequence may comprise about 17 nucleotides. In some cases, the polynucleotide sequence may comprise about 18 nucleotides. In some cases, the polynucleotide sequence may comprise about 19 nucleotides. In some cases, the polynucleotide sequence may comprise about 20 nucleotides. In some cases, the polynucleotide sequence may comprise about 21 nucleotides. In some cases, the polynucleotide sequence may comprise about 22 nucleotides. In some cases, the polynucleotide sequence may comprise about 23 nucleotides.
  • the polynucleotide sequence may comprise about 24 nucleotides. In some cases, the polynucleotide sequence may comprise about 25 nucleotides. In some cases, the polynucleotide sequence may comprise about 26 nucleotides. In some cases, the polynucleotide sequence may comprise about 27 nucleotides. In some cases, the polynucleotide sequence may comprise about 28 nucleotides. In some cases, the polynucleotide sequence may comprise about 29 nucleotides. In some cases, the polynucleotide sequence may comprise about 30 nucleotides. In some cases, the polynucleotide sequence may comprise about 31 nucleotides.
  • the polynucleotide sequence may comprise about 32 nucleotides. In some cases, the polynucleotide sequence may comprise about 33 nucleotides. In some cases, the polynucleotide sequence may comprise about 34 nucleotides. In some cases, the polynucleotide sequence may comprise about 35 nucleotides. In some cases, the polynucleotide sequence may comprise about 36 nucleotides. In some cases, the polynucleotide sequence may comprise about 37 nucleotides. In some cases, the polynucleotide sequence may comprise about 38 nucleotides. In some cases, the polynucleotide sequence may comprise about 39 nucleotides.
  • the polynucleotide sequence may comprise about 40 nucleotides. In some cases, the polynucleotide sequence may comprise about 41 nucleotides. In some cases, the polynucleotide sequence may comprise about 42 nucleotides. In some cases, the polynucleotide sequence may comprise about 43 nucleotides. In some cases, the polynucleotide sequence may comprise about 44 nucleotides. In some cases, the polynucleotide sequence may comprise about 45 nucleotides. In some cases, the polynucleotide sequence may comprise about 46 nucleotides. In some cases, the polynucleotide sequence may comprise about 47 nucleotides.
  • the polynucleotide sequence may comprise about 48 nucleotides. In some cases, the polynucleotide sequence may comprise about 49 nucleotides. In some cases, the polynucleotide sequence may comprise about 50 nucleotides. In some cases, the polynucleotide sequence may comprise about 51 nucleotides. In some cases, the polynucleotide sequence may comprise about 52 nucleotides. In some cases, the polynucleotide sequence may comprise about 53 nucleotides. In some cases, the polynucleotide sequence may comprise about 54 nucleotides. In some cases, the polynucleotide sequence may comprise about 55 nucleotides.
  • the polynucleotide sequence may comprise about 56 nucleotides. In some cases, the polynucleotide sequence may comprise about 57 nucleotides. In some cases, the polynucleotide sequence may comprise about 58 nucleotides. In some cases, the polynucleotide sequence may comprise about 59 nucleotides. In some cases, the polynucleotide sequence may comprise about 60 nucleotides. In some cases, the polynucleotide sequence may comprise about 61 nucleotides. In some cases, the polynucleotide sequence may comprise about 62 nucleotides. In some cases, the polynucleotide sequence may comprise about 63 nucleotides.
  • the polynucleotide sequence may comprise about 64 nucleotides. In some cases, the polynucleotide sequence may comprise about 65 nucleotides. In some cases, the polynucleotide sequence may comprise about 66 nucleotides. In some cases, the polynucleotide sequence may comprise about 67 nucleotides. In some cases, the polynucleotide sequence may comprise about 68 nucleotides. In some cases, the polynucleotide sequence may comprise about 69 nucleotides. In some cases, the polynucleotide sequence may comprise about 70 nucleotides. In some cases, the polynucleotide sequence may comprise about 71 nucleotides.
  • the polynucleotide sequence may comprise about 72 nucleotides. In some cases, the polynucleotide sequence may comprise about 73 nucleotides. In some cases, the polynucleotide sequence may comprise about 74 nucleotides. In some cases, the polynucleotide sequence may comprise about 75 nucleotides. In some cases, the polynucleotide sequence may comprise about 76 nucleotides. In some cases, the polynucleotide sequence may comprise about 77 nucleotides. In some cases, the polynucleotide sequence may comprise about 78 nucleotides. In some cases, the polynucleotide sequence may comprise about 79 nucleotides.
  • the polynucleotide sequence may comprise about 80 nucleotides. In some cases, the polynucleotide sequence may comprise about 81 nucleotides. In some cases, the polynucleotide sequence may comprise about 82 nucleotides. In some cases, the polynucleotide sequence may comprise about 83 nucleotides. In some cases, the polynucleotide sequence may comprise about 84 nucleotides. In some cases, the polynucleotide sequence may comprise about 85 nucleotides. In some cases, the polynucleotide sequence may comprise about 86 nucleotides. In some cases, the polynucleotide sequence may comprise about 87 nucleotides.
  • the polynucleotide sequence may comprise about 88 nucleotides. In some cases, the polynucleotide sequence may comprise about 89 nucleotides. In some cases, the polynucleotide sequence may comprise about 90 nucleotides. In some cases, the polynucleotide sequence may comprise about 91 nucleotides. In some cases, the polynucleotide sequence may comprise about 92 nucleotides. In some cases, the polynucleotide sequence may comprise about 93 nucleotides. In some cases, the polynucleotide sequence may comprise about 94 nucleotides. In some cases, the polynucleotide sequence may comprise about 95 nucleotides.
  • the polynucleotide sequence may comprise about 96 nucleotides. In some cases, the polynucleotide sequence may comprise about 97 nucleotides. In some cases, the polynucleotide sequence may comprise about 98 nucleotides. In some cases, the polynucleotide sequence may comprise about 99 nucleotides. In some cases, the polynucleotide sequence may comprise about 100 nucleotides.
  • the polynucleotide sequence may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more nucleotides.
  • the polynucleotide sequence may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides.
  • a reporter can comprise any numbers of nucleotides described thereof.
  • a reporter can be 5, 8, or 10 nucleotides in length.
  • a reporter can comprise any numbers of nucleotides described thereof.
  • a reporter can be about 10 nucleotides in length.
  • Reporters may comprise RNA. Reporters may comprise DNA. Reporters may also comprise both DNA and RNA. Reporters may be double-stranded. Reporters may be singlestranded. A reporter may comprise a single-stranded region. A reporter may comprise a doublestranded region. In some cases, reporters may comprise both single-stranded and doubles- stranded regions. In some instances, cleavage of the reporter produces, changes, or reduces a signal and thereby indicate the presence of the target nucleic acid in the sample. The systems and devices disclosed herein can be used to detect these signals, which can indicate whether a target nucleic acid is present in the sample.
  • the reporter can comprise a single-stranded nucleic acid sequence comprising at least one deoxyribonucleotide and at least one ribonucleotide.
  • the reporter can comprise a doublestranded nucleic acid sequence comprising at least one deoxyribonucleotide and at least one ribonucleotide.
  • the reporter can comprise a single-stranded nucleic acid sequence and a doublestranded nucleic acid region, each comprising at least one deoxyribonucleotide and at least one ribonucleotide.
  • the single-stranded region of a reporter may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more nucleotides.
  • the singlestranded region of the reporter may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides.
  • the single-stranded region may comprise about 5 to about 15 nucleotides.
  • the single-stranded region may comprise about 5 to about 20 nucleotides.
  • the single-stranded region may comprise about 5 to about 25 nucleotides.
  • the single-stranded region may comprise about 5 to about 50 nucleotides. In some cases, the single-stranded region may comprise about 5 to about 100 nucleotides. In some cases, the single-stranded region may comprise about 5 to about 200 nucleotides. In some cases, the single-stranded region may comprise about 5 to about 500 nucleotides. In some cases, the single-stranded region may comprise about 4 to about 15 nucleotides. In some cases, the single-stranded region may comprise about 3 to about 15 nucleotides. In some cases, the single-stranded region may comprise about 2 to about 15 nucleotides. In some cases, the single-stranded region may comprise about 1 to about 15 nucleotides.
  • the single-stranded region may comprise about 1 nucleotides. In some cases, the single-stranded region may comprise about 2 nucleotides. In some cases, the single-stranded region may comprise about 3 nucleotides. In some cases, the single-stranded region may comprise about 4 nucleotides. In some cases, the single-stranded region may comprise about 5 nucleotides. In some cases, the single-stranded region may comprise about 6 nucleotides. In some cases, the single-stranded region may comprise about 7 nucleotides. In some cases, the single-stranded region may comprise about 8 nucleotides. In some cases, the single-stranded region may comprise about 9 nucleotides.
  • the single-stranded region may comprise about 10 nucleotides. In some cases, the single-stranded region may comprise about 11 nucleotides. In some cases, the single-stranded region may comprise about 12 nucleotides. In some cases, the single-stranded region may comprise about 13 nucleotides. In some cases, the single-stranded region may comprise about 14 nucleotides. In some cases, the single-stranded region may comprise about 15 nucleotides. In some cases, the single-stranded region may comprise about 20 nucleotides. In some cases, the single-stranded region may comprise about 30 nucleotides. In some cases, the single-stranded region may comprise about 40 nucleotides.
  • the single-stranded region may comprise about 50 nucleotides. In some cases, the single-stranded region may comprise about 100 nucleotides. In some cases, the single-stranded region may comprise about 150 nucleotides. In some cases, the single-stranded region may comprise about 200 nucleotides. In some cases, the single-stranded region may comprise about 500 nucleotides.
  • the double-stranded region of a reporter may comprise at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
  • the double-stranded region of a reporter may comprise at most about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
  • nucleotide pairs 350, 400, 450, 500 or more nucleotide pairs.
  • the double-stranded region may comprise a length of about 10 nucleotides. In some cases, the double-stranded region may comprise a length of about 15 nucleotides. In some cases, the double-stranded region may comprise a length of about 20 nucleotides. In some cases, the double-stranded region may comprise a length of about 25 nucleotides. In some cases, the double-stranded region may comprise a length of about 30 nucleotides. In some cases, the double-stranded region may comprise a length of about 35 nucleotides. In some cases, the double-stranded region may comprise a length of about 40 nucleotides.
  • the double-stranded region may comprise a length of about 45 nucleotides. In some cases, the double-stranded region may comprise a length of about 50 nucleotides. In some cases, the double-stranded region may comprise a length of about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 60 nucleotides. In some cases, the double-stranded region may comprise a length of about 65 nucleotides. In some cases, the double-stranded region may comprise a length of about 70 nucleotides. In some cases, the double-stranded region may comprise a length of about 75 nucleotides.
  • the double-stranded region may comprise a length of about 80 nucleotides. In some cases, the double-stranded region may comprise a length of about 85 nucleotides. In some cases, the double-stranded region may comprise a length of about 90 nucleotides. In some cases, the double-stranded region may comprise a length of about 95 nucleotides. In some cases, the double-stranded region may comprise a length of about 100 nucleotides. In some cases, the double-stranded region may comprise a length of about 150 nucleotides. In some cases, the double-stranded region may comprise a length of about 200 nucleotides.
  • the double-stranded region may comprise a length of about 300 nucleotides. In some cases, the double-stranded region may comprise a length of about 400 nucleotides. In some cases, the double-stranded region may comprise a length of about 500 nucleotides.
  • the double-stranded region of a reporter may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90,
  • the doublestranded region of the reporter may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • the double-stranded region may comprise a length of about 45 to about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 40 to about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 35 to about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 30 to about 55 nucleotides.
  • the double-stranded region may comprise a length of about 25 to about 55 nucleotides. In some cases, the doublestranded region may comprise a length of about 20 to about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 15 to about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 10 to about 55 nucleotides. In some cases, the double-stranded region may comprise a length of about 45 to about 60 nucleotides. In some cases, the double-stranded region may comprise a length of about 45 to about 70 nucleotides.
  • the double-stranded region may comprise a length of about 45 to about 80 nucleotides. In some cases, the double-stranded region may comprise a length of about 45 to about 90 nucleotides. In some cases, the double-stranded region may comprise a length of about 45 to about 100 nucleotides. In some cases, the double-stranded region may comprise a length of about 45 to about 200 nucleotides. In some cases, the double-stranded region may comprise a length of about 45 to about 500 nucleotides. In some cases, the doublestranded region may comprise a length of about 5 to about 15 nucleotides.
  • the double-stranded region may comprise a length of about 5 to about 20 nucleotides. In some cases, the double-stranded region may comprise a length of about 5 to about 25 nucleotides. In some cases, the double-stranded region may comprise a length of about 5 to about 50 nucleotides. In some cases, the double-stranded region may comprise a length of about 5 to about 100 nucleotides. In some cases, the double-stranded region may comprise a length of about 5 to about 200 nucleotides. In some cases, the double-stranded region may comprise a length of about 5 to about 500 nucleotides.
  • the double-stranded region may comprise a length of about 4 to about 15 nucleotides. In some cases, the double-stranded region may comprise a length of about 3 to about 15 nucleotides. In some cases, the double-stranded region may comprise a length of about 2 to about 15 nucleotides. In some cases, the double-stranded region may comprise a length of about 1 to about 15 nucleotides.
  • the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 35 nucleotides. In some cases, the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 40 nucleotides. In some cases, the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 45 nucleotides. In some cases, the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 50 nucleotides.
  • the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 55 nucleotides. In some cases, the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 60 nucleotides. In some cases, the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 65 nucleotides. In some cases, the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 35 nucleotides.
  • the single-stranded region may comprise about 1 nucleotide, and the double-stranded region may comprise about 40 nucleotides. In some cases, the single-stranded region may comprise about 5 nucleotides, and the double-stranded region may comprise about 45 nucleotides. In some cases, the single-stranded region may comprise about 5 nucleotides, and the double-stranded region may comprise about 50 nucleotides. In some cases, the singlestranded region may comprise about 5 nucleotides, and the double-stranded region may comprise about 55 nucleotides.
  • the single-stranded region may comprise about 5 nucleotides, and the double-stranded region may comprise about 60 nucleotides. In some cases, the single-stranded region may comprise about 5 nucleotides, and the double-stranded region may comprise about 65 nucleotides. In some cases, the single-stranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 35 nucleotides. In some cases, the single-stranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 40 nucleotides.
  • the single-stranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 45 nucleotides. In some cases, the single-stranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 50 nucleotides. In some cases, the single-stranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 55 nucleotides. In some cases, the single-stranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 60 nucleotides.
  • the singlestranded region may comprise about 9 nucleotides, and the double-stranded region may comprise about 65 nucleotides. In some cases, the single-stranded region may comprise about 15 nucleotides, and the double-stranded region may comprise about 35 nucleotides. In some cases, the single-stranded region may comprise about 15 nucleotides, and the double-stranded region may comprise about 40 nucleotides. In some cases, the single-stranded region may comprise about 15 nucleotides, and the double-stranded region may comprise about 45 nucleotides.
  • the single-stranded region may comprise about 15 nucleotides, and the doublestranded region may comprise about 50 nucleotides. In some cases, the single-stranded region may comprise about 15 nucleotides, and the double-stranded region may comprise about 55 nucleotides. In some cases, the single-stranded region may comprise about 15 nucleotides, and the double-stranded region may comprise about 60 nucleotides. In some cases, the singlestranded region may comprise about 15 nucleotides, and the double-stranded region may comprise about 65 nucleotides.
  • the single-stranded region may comprise about 20 nucleotides, and the double-stranded region may comprise about 35 nucleotides. In some cases, the single-stranded region may comprise about 20 nucleotides, and the double-stranded region may comprise about 40 nucleotides. In some cases, the single-stranded region may comprise about 20 nucleotides, and the double-stranded region may comprise about 45 nucleotides. In some cases, the single-stranded region may comprise about 20 nucleotides, and the doublestranded region may comprise about 50 nucleotides.
  • the single-stranded region may comprise about 20 nucleotides, and the double-stranded region may comprise about 55 nucleotides. In some cases, the single-stranded region may comprise about 20 nucleotides, and the double-stranded region may comprise about 60 nucleotides. In some cases, the singlestranded region may comprise about 20 nucleotides, and the double-stranded region may comprise about 65 nucleotides.
  • the reporter comprises a nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the reporter comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between nonribonucleotide residues. In some cases, the reporter has only ribonucleotide residues.
  • the reporter compirses a nucleic acid comprising at least one deoxyribonucleotide residue at an internal position that functions as a cleavage site.
  • the reporter comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribonucleotide residues at an internal position.
  • the reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 deoxyribonucleotide residues at an internal position.
  • the deoxyribonucleotide residues are continuous.
  • the deoxyribonucleotide residues may be interspersed in between non-deoxyribonucleotide residues.
  • the reporter has only deoxyribonucleotide residues.
  • the reporter has only deoxyribonucleotide residues.
  • the reporter comprises nucleotides resistant to cleavage by the programmable nuclease described herein.
  • the reporter comprises synthetic nucleotides.
  • the reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue.
  • the reporter comprises at least one uracil ribonucleotide.
  • the reporter comprises at least two uracil ribonucleotides. Sometimes the reporter has only uracil ribonucleotides.
  • the reporter comprises at least one adenine ribonucleotide.
  • the reporter comprises at least two adenine ribonucleotides. In some cases, the reporter has only adenine ribonucleotides. In some cases, the reporter comprises at least one cytosine ribonucleotide. In some cases, the reporter comprises at least two cytosine ribonucleotides. In some cases, the reporter comprises at least one guanine ribonucleotide. In some cases, the reporter comprises at least two guanine ribonucleotides.
  • a reporter can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. A reporter can comprise a combination of modified and unmodified ribonucleotides and/or deoxyribonucleotides.
  • a reporter molecule comprises a single stranded nucleic acid comprising a detection moiety, wherein the nucleic acid of the reporter molecule is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal.
  • the reporter molecule comprises a single-stranded nucleic acid sequence comprising ribonucleotides.
  • the reporter molecule comprises a single-stranded nucleic acid sequence comprising deoxyribonucleotides.
  • the reporter molecule comprises a single-stranded nucleic acid sequence comprising deoxyribonucleotides and ribonucleotides.
  • nucleic acid sequences can be detected using a programmable RNA nuclease, a programmable DNA nuclease, or a combination thereof, as disclosed herein.
  • the programmable nuclease can be activated and cleave the reporter molecule upon binding of a guide nucleic acid to a target nucleic acid.
  • different compositions of reporter molecules can allow for multiplexing using different programmable nucleases (e.g., a programmable RNA nuclease and a programmable DNA nuclease).
  • the reporter may comprise any design detailed in Table 2 below or described herein.
  • the reporter may comprise a nucleic acid sequence at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100 % identical to any one of the sequences described in Table 2.
  • the reporter may comprise a nucleic acid sequence at least about 50 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 55 % identical to any one of SEQ IDs NO: 62- 67.
  • the reporter may comprise a nucleic acid sequence at least about 60 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 65 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 70 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 75 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 80 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 85 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 90 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 95 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 96 % identical to any one of SEQ IDs NO: 62- 67.
  • the reporter may comprise a nucleic acid sequence at least about 97 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 98 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence at least about 99 % identical to any one of SEQ IDs NO: 62-67.
  • the reporter may comprise a nucleic acid sequence of any one of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 50 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 55 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 60 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 65 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 70 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 75 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 80 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 85 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 90 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 95 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 96 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 97 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 98 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 99 % identical to any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences of any two of SEQ IDs NO: 62-67.
  • the reporter may comprise at least two nucleic acid sequences at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 50 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 55 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 60 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 65 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 70 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 75 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 80 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 85 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 90 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 95 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 96 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 97 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 98 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences at least about 99 % identical to SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter may comprise at least two nucleic acid sequences of SEQ ID NOs: 62, 63, 68, and 69.
  • the reporter molecule comprises a detection moiety capable of generating a detectable signal.
  • a signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric.
  • Suitable detectable labels and/or moieties that may provide a signal include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair, a fluorophore, a fluorescent protein, a quantum dot, and the like.
  • a detection moiety can be located at an end of a reporter. In some cases, a detection moiety can be located at an end of a nucleic acid of a reporter. Cleavage of the nucleic acid can release the detection moiety, thereby decreasing the signal a reporter. In some cases, a detection moiety can be located at the 3’ end of a nucleic acid of a reporter. For example, a fluorophore can be located at the 3’ end of a reporter, as shown in FIGs. 40A, 40C, or 40E. In other cases, the fluorophore can be at the 5’ end of a reporter.
  • a quenching moiety is on the other side of the cleavage site.
  • the quenching moiety is a fluorescence quenching moiety.
  • the reporter may comprise an immobilization moiety, a first nucleic acid, a fluorophore, a second nucleic acid, and a quencher moiety, as shown in FIGs. 40B or 40D.
  • the quencher moiety may quench the signal of the fluorophore. Cleavage of the second nucleic acid may release the quencher moiety, thereby increasing the signal of the reporter.
  • the quenching moiety is 5’ to the cleavage site and the fluorophore is 3’ to the cleavage site. In some cases, the fluorophore is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the reporter molecule. Sometimes the fluorophore is at the 3’ terminus of the reporter molecule. In some cases, the fluorophore is at the 5’ terminus of the reporter molecule. In some cases, the quenching moiety is at the 3’ terminus of the reporter molecule.
  • a substrate and/or detection region may comprise multiple reporters, multiple guide nucleic acids, or a combination thereof that are immobilized, dried, or otherwise deposited thereto. Localizing the guide nucleic acids and reporter may localize the detectable signal for each target nucleic acid to the detection spot, thus enabling the spatial multiplexing.
  • the detection region may comprise an array of detection spots at discrete locations. Each detection spot of the array may comprise an immobilized reporter and a different immobilized guide nucleic acid which is complementary to a different target nucleic acid of a plurality of target nucleic acids.
  • the immobilized reporter is cleaved by a complex comprising the programmable nuclease and the different immobilized guide nucleic acid to generate a different signal of a plurality of signals.
  • Each different signal may therefore be indicative of the presence or absence of a different target nucleic acid.
  • the target nucleic acids may be freely available within the fluid volume of the detection region.
  • the array may comprise a number of spots within a range of about 1 to about 200, within a range of about 3 to about 200, or within a range of about 10 to about 200.
  • the array may comprise at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
  • multiple guide nucleic acids for a single target nucleic acid may be combined within a single detection spot in order to increase a rate of reaction.
  • a substrate may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • a detection region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • each detection location may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more reporters and/or guide nucleic acids immobilized thereto.
  • the reagents described herein can also include buffers, which are compatible with the devices, systems, fluidic devices, kits, and methods disclosed herein.
  • the buffers described herein are compatible for use in the devices described herein and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether the target nucleic acid is in the sample (e.g., DETECTR reactions).
  • compositions disclosed herein e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof
  • buffers are compatible with the other reagents, samples, and support mediums as described herein for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry.
  • the methods described herein can also include the use of buffers, which are compatible with the methods disclosed herein.
  • a buffer may comprise HEPES, MES, TCEP, EGTA, Tween 20, KC1, MgCl 2 , glycerol, TIPP, or any combination thereof.
  • a buffer may comprise Tris-HCl pH 8.8, VLB, EGTA, CH3COOH, TCEP, IsoAmp, (NH4)2SO4, KC1, MgSO4, Tween20, KO Ac, MgOAc, BSA, or any combination thereof.
  • the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8.
  • the buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KC1.
  • the buffer may comprise 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl 2 .
  • the buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol.
  • the buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% glycerol.
  • the buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM Tris-HCl pH 8.8.
  • the buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KO Ac.
  • the buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM MgOAc.
  • the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM EGTA.
  • the buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% Tween 20.
  • a programmable nuclease-based assay is referred to as a DETECTR assay.
  • one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule).
  • a programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter.
  • a programmable nuclease detection (directly or indirectly) of reporter cleavage by a programmable nuclease to determine the presence of a target nucleic acid sequence may be referred to as 'DETECTR'.
  • the programmable nuclease can, in some cases, be referred to as an RNA-activated programmable RNA nuclease.
  • the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter.
  • a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease.
  • a programmable nuclease as described herein can be activated by a target RNA or a target DNA.
  • a programmable nuclease e.g., a Cas enzyme
  • the Cas enzyme can bind to a target ssDNA which initiates trans cleavage of RNA reporters.
  • a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA-activated programmable DNA nuclease.
  • the programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity.
  • Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety.
  • the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal.
  • the reporter and/or the detection moiety can be immobilized, dried, or otherwise deposited on a support medium.
  • the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid.
  • the detection moiety binds to a capture molecule on the support medium to be immobilized.
  • the detectable signal can be visualized on the support medium to assess the presence or concentration of one or more target nucleic acids associated with an ailment, such as a disease, cancer, or genetic disorder.
  • the devices, systems, fluidic devices, kits, and methods for detecting the presence of a target nucleic acid in a sample described herein may comprise a generation of a signal indicative of the presence or absence of the target nucleic acid in the sample.
  • the generation of a signal indicative of the presence or absence of the target nucleic acid in the sample as described herein is compatible with the methods and devices described herein and may result from the use of compositions disclosed herein (e.g.
  • detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
  • detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
  • the conjugate may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer.
  • the surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety or another affinity molecule of the cleaved detector molecule as described herein.
  • the detecting steps disclosed herein may involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, and/or measuring a signal indicating that the target nucleic acid is absent in a sample.
  • a signal is generated upon cleavage of the reporter by the programmable nuclease.
  • the signal changes upon cleavage of the reporter by the programmable nuclease.
  • a signal may be present in the absence of reporter cleavage and disappear or reduce upon cleavage of the target nucleic acid by the programmable nuclease.
  • a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
  • the workflow method may comprise: (1) sample collection from the patient and delivery to the device, (2) optional lysis, (3) optional amplification of the target nucleic acids, and (4) detection/readout.
  • amplification and detection are carried out in one reaction volume and is referred to as a one-pot reaction.
  • sample amplification is carried in a first reaction volume and detection is carried in a second reaction volume.
  • two-pot reactions are referred to as two-pot reactions.
  • carrying out multiple reactions in multiple reaction volumes is referred to as a multi-pot reaction.
  • Exemplary embodiments for one-pot, two-pot, and multi-pot reactions can be found in: PCT/US2021/033271, PCT/US2021/035031, PCT/US2021/063405, PCT/US2021/063844, PCT/US2022/034596, PCT/US2022/081842, and PCT/US2022/034110, all of which are herein incorporated by reference in their entireties.
  • the running of a programmable nuclease-based assay is referred to as a detection step.
  • the detection step may be preceded or coincide with nucleic acid amplification
  • nucleic acid amplification may comprise PCR.
  • nucleic acid amplification may comprise Loop-Mediated Isothermal Amplification (LAMP).
  • nucleic acid amplification and detection in a single reaction volume Any of the devices described herein may be configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In certain instances, methods include simultaneous amplification and detection in the same volume. In certain instances, methods include sequential amplification and detection in the same volume. In certain instances, methods include simultaneous amplification and detection in the same volume. In some embodiments, nucleic acid amplification may comprise LAMP amplification or a variation thereof. In some embodiments, nucleic acid amplification may comprise PCR amplification or a variation thereof.
  • the limit of detection for a programmable nuclease-based assay is 1 copy of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is 10 copies of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is from 1 to 10 copies of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is from 10 to 20 copies of a target nucleic acid per reaction.
  • the limit of detection for a programmable nuclease-based assay is from 20 to 30 copies of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is from 30 to 40 copies of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is from 40 to 50 copies of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is from 50 to 100 copies of a target nucleic acid per reaction.
  • the limit of detection for a programmable nuclease- based assay is from 100 to 1000 copies of a target nucleic acid per reaction. In some embodiments, the limit of detection for a programmable nuclease-based assay is greater than 1000 copies of a target nucleic acid per reaction.
  • Programmable nuclease-based diagnostic reactions are generally performed in solution where the programmable nuclease-guide nucleic acid complexes can freely bind target molecules and reporters.
  • reactions where all components are in solution can limit the designs of nucleic acid diagnostic assays, especially in microfluidic devices.
  • a system where various components of the programmable nuclease-based diagnostic reaction are immobilized on a surface may enable designs where multiple readouts can be accomplished within a single reaction chamber and/or in a single cartridge.
  • One or more components or reagents of a programmable nuclease-based detection reaction may be suspended in solution or immobilized on a surface.
  • Programmable nucleases, guide nucleic acids, and/or reporters may be suspended in solution or immobilized on a surface.
  • the reporter, programmable nuclease, and/or guide nucleic acid can be immobilized on the surface of a chamber in a device as disclosed herein.
  • the reporter, programmable nuclease, and/or guide nucleic acid can be immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet placed below the chamber.
  • An immobilized programmable nuclease can be capable of being activated and cleaving a free-floating or immobilized reporter.
  • An immobilized guide nucleic acid can be capable of binding a target nucleic acid and activating a programmable nuclease complexed thereto.
  • An immobilized reporter can be capable of being cleaved by the activated programmable nuclease, thereby generating a detectable signal.
  • Any of the devices described herein may comprise one or more immobilized detection reagent components (e.g., programmable nuclease, guide nucleic acid, and/or reporter).
  • methods include immobilization of programmable nucleases (e.g., Cas proteins or Cas enzymes), reporters, and/or guide nucleic acids (e.g., gRNAs).
  • various programmable nuclease-based diagnostic reaction components are modified with biotin.
  • these biotinylated programmable nuclease-based diagnostic reaction components are tested for immobilization on surfaces coated with streptavidin.
  • the biotin-streptavidin interaction is used as a model system for other immobilization chemistries.
  • NHS-Amine chemistry is used for immobilization of programmable nuclease-based reaction components.
  • amino modifications are used for immobilization of programmable nuclease-based reaction components.
  • maleimide-thiol chemistry is used for immobilization of programmable nuclease-based reaction components.
  • epoxy-amine chemistry is used for immobilization of programmable nuclease-based reaction components.
  • hydrogels are used for immobilization of programmable nuclease-based reaction components.
  • chemical modifications of amino acid residues in the Cas protein enable attachment to a surface.
  • guide nucleic acids are immobilized by adding various chemical modifications at the 5’ or 3’ end of the guide nucleic acids that are compatible with a selected surface chemistry.
  • fluorescence-quenching (FQ), or other reporter detection moiety chemistries are attached to surfaces using similar chemical modifications as gRNAs.
  • these attached reporters are cleaved by a programmable nuclease, which leads to either activated molecules (e.g., detection moieties) that remain attached to the surface or activated molecules that are released into solution.
  • the programmable nuclease complex may be immobilized by a guide nucleic acid and cleave surrounding fluorophore-quencher reporters that are also immobilized to a surface.
  • the quencher may be released into solution, leaving a localized fluorescent signal.
  • the fluorophore may be released into solution, thereby generating a fluorescent signal in solution.
  • the programmable nuclease, guide nucleic acid, reporter, or a combination thereof can be immobilized to a device surface (e.g., by a linkage).
  • the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, an interaction (e.g., a covalent or noncovalent bond) between members of a binding pair (e.g., streptavidin and biotin), an amide bond, or any combination thereof.
  • the linkage may be the same or different for each species.
  • the guide nucleic acid may be immobilized to the surface by a singlestranded linker polynucleotide
  • the reporters may be immobilized by the interaction between a first member of a binding pair on the reporters and a second member of a binding pair on the surface.
  • binding pair refers to a first and a second moiety that have a specific binding affinity for each other.
  • a binding pair has a dissociation constant Kd of less than or equal to about: 10' 8 mol/L, 10' 9 mol/L, IO' 10 mol/L, 10' 11 mol/L, 10' 12 mol/L, 10' 13 mol/L, 10' 14 mol/L, 10' 15 mol/L, or ranges including two of these values as endpoints.
  • Non limiting examples of binding pairs include an antibody or an antigen-binding portion thereof and an antigen (e.g., fluorescein, digoxin, digoxigenin); a biotin (bio) moiety and an avidin (or streptavidin) moiety; a dinitrophenol (DNP) and an anti-DNP antibody; a hapten and an anti hapten; folate and a folate binding protein; vitamin B 12 and an intrinsic factor; a carbohydrate and a lectin or carbohydrate receptor; a polysaccharide and a polysaccharide binding moiety; a lectin and a receptor; a ligand and a receptor; a drug and a drug receptor; complementary chemical reactive groups (e.g., sulfhydryl/maleimide, thiol/maleimide, sulfhydryl/haloacetyl derivative, amine/epoxy, amine/isotriocyanate, amine/succinimidyl este
  • the linkage utilizes non-specific absorption.
  • the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface.
  • the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface.
  • the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5’ end of the guide nucleic acid and the surface.
  • the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3’ end of the guide nucleic acid and the surface.
  • the programmable nuclease, guide nucleic acid, and/or the reporter are immobilized to or within a polymer matrix.
  • the polymer matrix may comprise a hydrogel. Co-polymerization of the programmable nuclease, guide nucleic acid, or the reporter into the polymer matrix may result in a higher density of reporter/unit volume or reporter/unit area than other immobilization methods utilizing surface immobilization (e.g., onto beads, after matrix polymerization, etc.).
  • Co-polymerization of the programmable nuclease, guide nucleic acid, or the reporter into the polymer matrix may result in less undesired release of the reporter (e.g., during an assay, a measurement, or on the shelf), and thus may cause less background signal, than other immobilization strategies (e.g., conjugation to a pre-formed hydrogel, bead, etc.). In at least some instances this may be due to better incorporation of reporters into the polymer matrix as a co-polymer and fewer “free” reporter molecules retained on the hydrogel via non-covalent interactions or non-specific binding interactions.
  • a plurality of oligomers and a plurality of polymerizable oligomers may comprise an irregular or non-uniform mixture.
  • the irregularity of the mixture of polymerizable oligomers and unfunctionalized oligomers may allow pores to form within the hydrogel (i.e., the unfunctionalized oligomers may act as a porogen).
  • the irregular mixture of oligomers may result in phase separation during polymerization that allows for the generation of pores of sufficient size for free-floating programmable nucleases to diffuse into the hydrogel and access immobilized internal reporter molecules.
  • the relative percentages and/or molecular weights of the oligomers may be varied to vary the pore size of the hydrogel. For example, pore size may be tailored to increase the diffusion coefficient of the programmable nucleases.
  • the functional groups attached to the reporters and/or guide nucleic acids may be selected to preferentially incorporate the reporters and/or guide nucleic acids into the polymer matrix via covalent binding at the functional group versus other locations along the nucleic acid backbone of the reporter and/or guide nucleic acid.
  • the functional groups attached to the reporters and/or guide nucleic acids may be selected to favorably transfer free radicals from the functionalized ends of polymerizable oligomers to the functional group on the end of the reporter and/or guide nucleic acid (e.g., 5’ end), thereby forming a covalent bond and immobilizing the reporter and/or guide nucleic acid rather than destroying other parts of the reporter and/or guide nucleic acid molecules, respectively.
  • the functional group may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5’ thiol modifier, a 3’ thiol modifier, an amine group, a I-LinkerTM group, methacryl group, or any combination thereof.
  • a variety of functional groups may be used depending on the desired properties of the immobilized components.
  • Methods consistent with the present disclosure include a multiplexing method of assaying for a plurality of target nucleic acids in a sample.
  • a multiplexing method may comprise a) contacting the sample to a programmable nuclease complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and b) assaying for a signal indicating cleavage of at least some reporters (e.g., protein- nucleic acids) of a population of reporter molecules (e.g., protein-nucleic acids), wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample.
  • reporters e.g., protein- nucleic acids
  • reporter molecules
  • Multiplexing can comprise spatial multiplexing wherein multiple different target nucleic acids are detected at the same time, but the reactions are spatially separated.
  • the multiple target nucleic acids are detected using the same programmable nuclease, but different guide nucleic acids.
  • the multiple target nucleic acids sometimes are detected using different programmable nucleases.
  • the method may involve using a first programmable nuclease, which upon activation (e.g., after binding of a first guide nucleic acid to a first target), cleaves a nucleic acid of a first reporter and using a second programmable nuclease, which upon activation (e.g., after binding of a second guide nucleic acid to a second target) cleaves a nucleic acid of a second reporter.
  • Spatially separated reactions may, for example, occur within an array comprising a plurality of different detection locations each comprising one or more immobilized detection reaction component(s) thereon.
  • spatially separated reactions may occur in different wells of a detection region, e.g., within different microwells, each microwell containing different detection reaction components configured to detect different target nucleic acids.
  • multiplexing can be single reaction multiplexing, wherein multiple different target acids are detected in a single reaction volume. Often, at least two different programmable nucleases are used in single reaction multiplexing.
  • multiplexing can be enabled by immobilization of multiple categories of detector nucleic acids within a fluidic system, to enable detection of multiple target nucleic acids within a single fluidic system. Multiplexing allows for detection of multiple target nucleic acids in one kit or system.
  • the multiple target nucleic acids comprise different target nucleic acids from a virus, a bacterium, or a pathogen responsible for one disease.
  • the multiple target nucleic acids comprise different target nucleic acids associated with a cancer or genetic disorder.
  • Multiplexing for one disease, cancer, or genetic disorder increases at least one of sensitivity, specificity, or accuracy of the assay to detect the presence of the disease in the sample.
  • the multiple target nucleic acids comprise target nucleic acids directed to different viruses, bacteria, or pathogens responsible for more than one disease.
  • multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes of the same bacteria or pathogen responsible for a disease, for example, for a wildtype genotype of a bacteria or pathogen and for genotype of a bacteria or pathogen comprising a mutation, such as a single nucleotide polymorphism (SNP) that can confer resistance to a treatment, such as antibiotic treatment.
  • SNP single nucleotide polymorphism
  • multiplexing allows for multiplexed detection of multiple genomic alleles.
  • multiplexing may comprise method of assaying comprising a single assay for a microorganism species using a first programmable nuclease and an antibiotic resistance pattern in a microorganism using a second programmable nuclease.
  • multiplexing allows for discrimination between multiple target nucleic acids of different pathogen strains, for example, HP VI 6 and HP VI 8.
  • multiplexing allows for discrimination between multiple target nucleic acids of different variants of a pathogen, for example, alpha and delta SARS-CoV-2 variants.
  • the multiple target nucleic acids comprise target nucleic acids directed to different cancers or genetic disorders.
  • multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes, for example, for a wild-type genotype and for SNP genotype.
  • Multiplexing for multiple diseases, cancers, or genetic disorders provides the capability to test a panel of diseases from a single sample. For example, multiplexing for multiple diseases can be valuable in a broad panel testing of a new patient or in epidemiological surveys. Often multiplexing is used for identifying bacterial pathogens in sepsis or other diseases associated with multiple pathogens.
  • a method of quantification for a disease panel may comprise assaying for a plurality of unique target nucleic acids in a plurality of aliquots from a sample, assaying for a control nucleic acid control in a second aliquot of the sample, and quantifying a plurality of signals of the plurality of unique target nucleic acids by measuring signals produced by cleavage of detector nucleic acids compared to the signal produced in the second aliquot.
  • a unique target nucleic acid refers to the sequence of a nucleic acid that has an at least one nucleotide difference from the sequences of the other nucleic acids in the plurality.
  • a unique target nucleic population can comprise multiple copies of the unique target nucleic acid.
  • the plurality of unique target nucleic acids is from a plurality of bacterial pathogens in the sample.
  • the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 2 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 3 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 4 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 5 different target nucleic acids in a single reaction.
  • the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single reaction. In some instances, the multiplexed kits detect at least 2 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 3 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 4 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 5 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single kit.
  • the multiplexed kits detect from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 9, from 3 to 9, from 4 to 9, from 5 to 9, from 6 to 9, from 7 to 9, from 8 to 9, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 7, from 3 to 7, from 4 to 7, from 5 to 7, from 6 to 7, from 2 to 6, from 3 to 6, from 4 to 6, from 5 to 6, from 2 to 5, from 3 to 5, from 4 to 5, from 2 to 4, from 3 to 4, or from 2 to 3 different target nucleic acids in a single kit.
  • Multiplexing can be carried out in a single-pot or “one-pot” reaction, where reverse transcription, amplification, in vitro transcription, or any combination thereof, and detection are carried out in a single volume. Multiplexing can be carried out in a “two-pot reaction”, where reverse transcription, amplification, in vitro transcription, or any combination thereof, are carried out in a first volume and detection is carried out in a second volume.
  • multiplexing can comprise detecting multiple targets with a single probe.
  • multiplexing can comprise detecting multiple targets with multiple probes.
  • the multiple probes can be configured to detect a presence of a particular sequence, target nucleic acid, or a plurality of different target sequences or nucleic acids.
  • any of the devices described herein may comprise one or more sample preparation reagents. Any of the devices described herein may comprise sample preparation reagents as dried reagents. Dried reagents may comprise solids and/or semi-solids. In certain instances, dried reagents may comprise lyophilized reagents. Any of the devices described herein may comprise one or more lyophilized reagents (e.g., amplification reagents, programmable nucleases, buffers, excipients, etc.). In certain instances, methods include sample lysis, concentration, and/or filtration. In certain instances, methods include reconstitution of one or more lyophilized reagents.
  • sample preparation reagents as dried reagents. Dried reagents may comprise solids and/or semi-solids. In certain instances, dried reagents may comprise lyophilized reagents. Any of the devices described herein may comprise one or more lyophilized reagents (e.g., amplification
  • lyophilized reagents may be in the form of lyophilized beads, spheres, and/or particulates.
  • the lyophilized bead, sphere, and/or particulate may comprise either single or multiple compounds.
  • the lyophilized bead, sphere, and/or particulate may be adjusted to various moisture levels or hygroscopy.
  • the lyophilized bead, sphere, and/or particulate may comprise assay internal standards.
  • the lyophilized bead, sphere, and/or particulate may have diameters between about 0.5 millimeters to about 5 millimeters in diameter.
  • a programmable nuclease-based detection e.g., DETECTR
  • Such embodiments allow for adapting the buffer for binding a substrate to perform a concentration step.
  • experiments may be performed to evaluate the lysis (sample is evaluated directly in the assay) and binding (the sample is eluted from magnetic beads) characteristics of buffers with different components.
  • the input sample is the same concentration as the eluted sample.
  • crude lysis buffer is used in a one-pot assay with Casl4a.l (SEQ ID NO: 3).
  • the enzyme having SEQ ID NO: 17 is used with the programmable nuclease-based detection assay.
  • a control study involving sample preparation optimization of the LANCR (multiplexed isothermal amplification) assay involves: 25 pL sample + 25 pL lysis buffer and incubation at 25C for 1 minute.
  • the LANCR reaction is run as follows: 5 pL sample in a 25 pL reaction volume (standard conditions).
  • the DETECTR reaction is run as follows: 2 pL LANCR product in 20 pL reaction volume (standard conditions). Sample: 250 copies/rxn SeraCare SARS-CoV-2 reference RNA.
  • Trehalose, Raffinose, PVP 40, sorbitol, Mannitol, Mannose, or a combination thereof are used for lyophilization.
  • Trehalose may be used to control the rate of the reaction.
  • programmable nuclease-based assays utilize a Casl2 protein, a Cast 3 protein, a Cast 4 protein, or a CasPhi protein.
  • amplification e.g., RT-LAMP
  • programmable nuclease-based detection e.g., DETECTR
  • master mixes of reagents are lyophilized in the same combined master mix.
  • amplification and programmable nuclease-based detection master mixes of reagents and target may be lyophilized in the same combined master mix.
  • one-pot refers to the combination of both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reaction reagents in one volume.
  • amplification e.g., RT-LAMP
  • detection e.g., DETECTR
  • an excipient is used to confirm reagent stability throughout the lyophilization process, comprising freezing and drying steps.
  • excipients are sugars.
  • a master mix of assay reagents may be reconstituted after lyophilization.
  • a master mix of DETECTR assay reagents may be reconstituted after lyophilization.
  • a master mix of DETECTR assay reagents, including a Cast 2 protein for example may be reconstituted after lyophilization.
  • a master mix of amplification and programmable nuclease-based detection (e.g., DETECTR) assay reagents, including a Casl2 for example may be reconstituted after lyophilization.
  • a master mix of amplification e.g., RT-LAMP
  • programmable nuclease-based detection e.g., DETECTR
  • assay reagents including a Casl2 protein
  • the master mix of reagents and target for one assay is lyophilized. In some embodiments, the master mixes from more than one assay may be pooled and lyophilized.
  • lyophilized master mixes of reagents from more than one assay may be prepared in volumes of less than 1 mL. In some embodiments, lyophilized master mixes of reagents from more than one assay may be prepared in volumes of less than 250 pL. In some embodiments, lyophilized master mixes of reagents from more than one assay may be prepared in volumes of less than 25 pL. In some embodiments, lyophilized master mixes of reagents from more than one assay may be prepared in volumes of less than 10 pL.
  • an excipient is used to stabilize the sample throughout the lyophilization process that may comprise freezing and drying steps.
  • a viral, bacterial, and/or high temperature inactivator may be used.
  • programmable nuclease-based assays may comprise one or more controls.
  • the assay controls may comprise non-specific binding controls.
  • RNase may be used as a control against the programmable nuclease for cleavage activity.
  • DNase may be used as a control against the programmable nuclease for cleavage activity.
  • an assay control may comprise a no-target-control (NTC), wherein a particular control sample does not contain at least one target.
  • a control sample may comprise target nucleic acids added to the control sample at known concentrations.
  • the method can comprise collecting a sample.
  • the sample can comprise any type of sample as described herein.
  • the method can comprise preparing the sample.
  • Sample preparation can comprise one or more sample preparation steps.
  • the one or more sample preparation steps can be performed in any suitable order.
  • the one or more sample preparation steps can comprise physical filtration of non-target materials using a macro filter.
  • the one or more sample preparation steps can comprise nucleic acid purification.
  • the one or more sample preparation steps can comprise nucleic acid concentration.
  • the one or more sample preparation steps can comprise lysis.
  • the one or more sample preparation steps can comprise heat inactivation.
  • the one or more sample preparation steps can comprise chemical inactivation.
  • the one or more sample preparation steps can comprise neutralization.
  • the one or more sample preparation steps can comprise adding one or more enzymes or reagents to prepare the sample for target detection.
  • the method can comprise generating one or more droplets, aliquots, volumes, or subsamples from the sample.
  • the one or more droplets, aliquots, volumes, or subsamples can correspond to a volumetric portion of the sample.
  • the sample can be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more droplets, aliquots, volumes, or subsamples. In some embodiments, the sample is not divided into subsamples.
  • the method can comprise amplifying one or more targets within the sample.
  • the method can comprise amplifying one or more targets within each droplet, aliquot, volumes, or subsample. Amplification of the one or more targets within each droplet or volume can be performed in parallel and/or simultaneously for each droplet or volume. Dividing the sample into a plurality of droplets or volumes can enhance a speed and/or an efficiency of the amplification process (e.g., a thermocycling process) since the droplets comprise a smaller volume of material than the bulk sample introduced.
  • amplification process e.g., a thermocycling process
  • Amplifying the one or more targets within each individual droplet or volume can also permit effective amplification of various target nucleic acids that cannot be amplified as efficiently in a bulk sample containing the various target nucleic acids if the bulk sample were to undergo a singular amplification process.
  • amplification is performed on the bulk sample without first dividing the sample into subsamples.
  • the method can further comprise using a CRISPR-based or programmable nuclease- based detection module to detect one or more targets (e.g., target sequences or target nucleic acids) in the sample.
  • targets e.g., target sequences or target nucleic acids
  • the sample can be divided into a plurality of droplets, aliquots, volumes, or subsamples to facilitate sample preparation and to enhance the detection capabilities of the devices of the present disclosure.
  • the sample can be provided manually to the detection device of the present disclosure.
  • a swab sample can be dipped into a solution and the sample/solution can be pipetted into the device.
  • the sample can be provided via an automated syringe.
  • the automated syringe can be configured to control a flow rate at which the sample is provided to the detection device.
  • the automated syringe can be configured to control a volume of the sample that is provided to the detection device over a predetermined period.
  • the sample can be provided directly to the detection device of the present disclosure.
  • a swab sample can be inserted into a sample receiver and/or a sample chamber on the detection device.
  • the sample can be prepared before one or more targets are detected within the sample.
  • the sample preparation steps described herein can process a crude sample to generate a pure or purer sample.
  • Sample preparation can comprise one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and/or elution.
  • sample preparation can comprise the following steps, in any order, including sample collection, nucleic acid purification, heat inactivation, neutralization, and/or base/acid lysis.
  • nucleic acid purification can be performed on the sample. Purification can comprise disrupting a biological matrix of a cell to release nucleic acids, denaturing structural proteins associated with the nucleic acids (nucleoproteins), inactivating nucleases that can degrade the isolated product (e.g., RNase and/or DNase), and/or removing contaminants (e.g., proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, and/or other cellular debris).
  • nucleic acid purification may involve liquid-liquid extraction, solid-liquid extraction, and/or solid-phase extraction techniques.
  • Solid-phase extraction may be preferred for smaller sample volumes and/or improved cartridge-based performance (e.g., by reducing solvent volumes and/or simpler workflow automation).
  • Solid-phase nucleic acid purification may utilize a silica-based method such as the Boom method.
  • silica particles e.g., silica beads or silica magnetic beads
  • glass fiber filters e.g., glass fiber filters, silica-coated membranes/meshes/filters, etc. may be used to capture nucleic acids from the sample prior to elution therefrom.
  • solid-phase nucleic acid purification may utilize charge switching for nucleic acid concentration.
  • particles e.g., beads or magnetic beads
  • surfaces e.g., membranes, meshes, filters, etc.
  • an ionizable coating such as chitosan.
  • the sample may be agitated (e.g., mixed, shaken, flown, sonicated, etc.) during purification and/or elution.
  • lysis of a collected sample can be performed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme, and/or a savinase).
  • a solution of reagents can be used to lyse the cells in the sample and release the nucleic acids so that they are accessible to the programmable nuclease.
  • Active ingredients of the solution can be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength, and pH. Chaotropic agents or chaotropes are substances that disrupt the three-dimensional structure in macromolecules such as proteins, DNA, or RNA.
  • One example protocol may comprise a 4 M guanidinium isothiocyanate, 25 mM sodium citrate.2H2O, 0.5% (w/v) sodium lauryl sarcosinate, and 0.1 M P-mercaptoethanol), but numerous commercial buffers for different cellular targets can also be used.
  • Alkaline buffers i.e., buffers with a pH above 7
  • the alkaline buffer has a pH of about or more than about 8, 8.5, 9, 9.5, 10, or higher.
  • acidic buffers i.e., buffers with a pH below 7 are used for lysis.
  • the alkaline buffer has a pH of about 10. Any suitable base may be used for the alkaline buffer, including organic or inorganic bases. In some embodiments, the base is sodium hydroxide. In some embodiments, the acidic buffer has a pH of about or less than about 6, 5.5, 5, 4.5, 4, or lower. In some embodiments, the acidic buffer has a pH of about 5. Any suitable acid may be used for the acidic buffer, including organic or inorganic acids. In some embodiments, the acid is acetic acid. Detergents such as sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB) can also be implemented to chemical lysis buffers.
  • SDS sodium dodecyl sulfate
  • CTAB cetyl trimethylammonium bromide
  • Cell lysis can also be performed by physical, mechanical, thermal, or enzymatic means, in addition to or instead of chemically-induced cell lysis mentioned previously.
  • nanoscale barbs, nanowires, acoustic generators (e.g., ultrasonic horns), integrated lasers, integrated heaters, and/or microcapillary probes can be used to perform lysis.
  • the sample may be agitated (e.g., mixed, shaken, flown, sonicated, etc.) during lysis.
  • lysis comprises exposure to both an acidic and an elevated temperature (e.g., 65 °C to 95 °C, 70 °C to 90 °C, or 75 °C to 85 °C).
  • inactivation can be performed on the sample to inactivate or neutralize conditions employed in a lysis step.
  • a processed/lysed sample can undergo heat inactivation to inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent).
  • the sample may be agitated (e.g., mixed, shaken, flown, sonicated, bubbled, etc.) during heat-inactivation or neutralization.
  • a heating element integrated into the detection device or disposed within the instrument can be used for heat-inactivation.
  • the heating element can be powered by a battery or another source of thermal or electric energy that is integrated with the detection device or instrument.
  • the heating element for heat-inactivation is the same as the heating element for lysis, amplification, and/or detection.
  • inactivation may comprise introducing a protease inhibitor.
  • inactivation may comprise a neutralization step that lowers the pH of the solution, such as to a pH of about 7, 7.5, 8, or 8.5.
  • inactivation may comprise a neutralization step that lowers the pH of the solution, such as to a pH of about 8.8.
  • inactivation may comprise a neutralization step that raises the pH of the solution, such as to a pH of about 7, 7.5, 8, or 8.5.
  • inactivation may comprise a neutralization step that raises the pH of the solution, such as to a pH of about 8.8.
  • Neutralization may utilize any suitable base (e.g., potassium hydroxide or Tris) or acid (e.g., potassium acetate), depending on the pH of the lysis buffer, which may be provided in any suitable form (e.g., pellet or concentrated solution).
  • a filtering step to remove debris may be included between lysis and inactivation, after inactivation, or both.
  • a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme.
  • the target nucleic acid within a purified sample can be amplified to generate a target amplicon.
  • amplification can be accomplished using loop mediated amplification (LAMP), isothermal recombinase polymerase amplification (RPA), and/or polymerase chain reaction (PCR).
  • LAMP loop mediated amplification
  • RPA isothermal recombinase polymerase amplification
  • PCR polymerase chain reaction
  • digital droplet amplification can used.
  • Such nucleic acid amplification of the sample can improve at least one of a sensitivity, specificity, or accuracy of the detection of the target nucleic acid.
  • the reagents for nucleic acid amplification can comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase.
  • the nucleic acid amplification can be transcription mediated amplification (TMA).
  • TMA transcription mediated amplification
  • Nucleic acid amplification can be helicase dependent amplification (HD A) or circular helicase dependent amplification (cHDA).
  • HD A helicase dependent amplification
  • cHDA circular helicase dependent amplification
  • SDA strand displacement amplification
  • the nucleic acid amplification can be recombinase polymerase amplification (RPA).
  • the nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).
  • the nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes.
  • the nucleic acid amplification is performed for from 1 to 60, from 5 to 55, from 10 to 50, from 15 to 45, from 20 to 40, or from 25 to 35 minutes.
  • the nucleic acid amplification is performed for from 5 to 60, from 10 to 60, from 15 to 60, from 30 to 60, from 45 to 60, from 1 to 45, from 5 to 45, from 10 to 45, from 30 to 45, from 1 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 1 to 15, from 5 to 15, or from 10 to 15 minutes.
  • amplification can comprise thermocycling of the sample.
  • Thermocycling can be carried out for one or more droplets or volumes of the sample in parallel and/or independently in separate locations. In some embodiments, this can be accomplished by methods such as (1) by holding droplets or volumes stationary in locations where a heating element is in close proximity to the droplet or volume on one of the droplet or volume sides and a heat sink element is in close proximity to the other side of the droplet or volume, or (2) flowing the droplet of volume through zones in a fluid channel where heat flows across it from a heating source to a heat sink.
  • one or more resistive heating elements can be used to perform thermocycling.
  • thermocycling may comprise one or more reactions at different temperatures. In some cases, the reactions can include an annealing reaction, a denaturation reaction, and/or an extension reaction.
  • an annealing temperature of the thermocycling reaction may be performed at a temperature around 45°C to 75°C.
  • the annealing temperature may be at a temperature of about 45 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 52 °C, about 54 °C, about 56 °C, about 58 °C, about 60 °C, about 62 °C, about 64 °C, about 66 °C, about 68 °C, about 70 °C, about 72 °C, about 74 °C, or about 76 °C.
  • a denaturation temperature of the thermocycling reaction may be performed at a temperature around 90°C to about 110°C.
  • the denaturation temperature may be at a temperature of about 90°C, about 91 °C, about 92°C, about 93 °C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, about 100°C, about 101°C, about 102°C, about 103°C, about 104°C, about 105°C, about 106°C, about 107°C, about 108°C, about 109°C, or about 110°C.
  • an extension temperature of the thermocycling reaction may be performed at a temperature from around 55°C to about 85°C.
  • the extension temperature may be at a temperature of about 55°C, about 57°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 68°C, about 70°C, about 71°C, about 72°C, about 73°C, about 75°C, about 76°C, about 78°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, or about 85°C.
  • amplification can comprise isothermal amplification of the sample.
  • Isothermal amplification ca be carried out for one or more droplets or volumes of the sample in parallel and/or independently in separate locations. In some embodiments, this can be accomplished by holding droplets or volumes stationary in locations where a heating element is in close proximity to the droplet or volume on one of the droplet or volume sides so as to maintain a constant temperature within the droplet or volume.
  • one or more resistive heating elements can be used to perform isothermal amplification.
  • the nucleic acid amplification reaction is performed at a temperature of around 20-65°C.
  • the nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
  • the nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
  • the nucleic acid amplification reaction is performed at a temperature of from 20°C to 45°C, from 25°C to 40°C, from 30°C to 40°C, or from 35°C to 40°C.
  • the nucleic acid amplification reaction is performed at a temperature of from 45°C to 65°C, from 50°C to 65°C, from 55°C to 65°C, or from 60°C to 65°C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 20 °C to 45 °C, from 25 °C to 45 °C, from 30 °C to 45 °C, from 35 °C to 45 °C, from 40 °C to 45 °C, from 20 °C to 37 °C, from 25 °C to 37 °C, from 30 °C to 37 °C, from 35 °C to 37 °C, from 20 °C to 30 °C, from 25 °C to 30 °C, from 20 °C to 25 °C, or from about 22 °C to 25 °C.
  • the nucleic acid amplification reaction can be performed at a temperature that ranges from about 40 °C to 65 °C, from 45 °C to 65 °C, from 50 °C to 65 °C, from 55 °C to 65 °C, from 60 °C to 65 °C, from 40 °C to 60 °C, from 45 °C to 60 °C, from 50 °C to 60 °C, from 55 °C to 60 °C, from 40 °C to 55 °C, from 45 °C to 55 °C, from 50 °C to 55 °C, from 40 °C to 50 °C, or from about 45 °C to 50 °C.
  • target nucleic acid can optionally be amplified before binding to the guide nucleic acid (e.g., crRNA) of the programmable nuclease complex (e.g., CRISPR enzyme).
  • This amplification can, for example, be PCR amplification or isothermal amplification.
  • This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target RNA.
  • the reagents for nucleic acid amplification can comprise a recombinase, a oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase.
  • SSB single-stranded DNA binding
  • the nucleic acid amplification can be transcription mediated amplification (TMA).
  • TMA transcription mediated amplification
  • Nucleic acid amplification can be helicase dependent amplification (HD A) or circular helicase dependent amplification (cHDA).
  • SDA strand displacement amplification
  • the nucleic acid amplification can be recombinase polymerase amplification (RPA).
  • RPA recombinase polymerase amplification
  • the nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR).
  • Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).
  • the nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes.
  • the nucleic acid amplification reaction is performed at a temperature of around 20- 45°C. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 45-65 °C.
  • the nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
  • the nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
  • a target nucleic acid within the sample can undergo reverse transcription before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme.
  • the target nucleic acid within a purified sample can be reverse transcribed.
  • reverse transcription can be accomplished using a reverse transcriptase.
  • Reverse transcription can be combined with any of the amplification techniques described herein.
  • a reverse transcription step may be performed at a temperature of around 45°C to about 75°C.
  • the reverse transcription may be at a temperature of about 45°C, about 47°C, about 48°C, about 49°C, about 50°C, about 52°C, about 54°C, about 55°C, about 57°C, about 59°C, about 60°C, about 61°C, about 63°C, about 65°C, about 66°C, about 68°C, about 70°C, about 72°C, about 73°C, or about 75°C.
  • the nucleic acid-based assay may be a programmable nuclease-based assay.
  • Systems may comprise any of the instruments and/or cartridges described herein.
  • the instrument and the cartridge may be configured to mate with one another to enable mechanical manipulation, activation, control, and/or communication between electronic circuits (e.g., in the cartridge and the instrument), thermal interfaces, ultrasonic interfaces, pneumatic interfaces, and/or optical interfaces.
  • the instrument may comprise a receptacle (e.g., an open slot or dock) configured (e.g., sized and shaped) to receive the cartridge.
  • the cartridge may be self-aligning with and/or locked within the instrument after insertion into its receptacle.
  • the selfalignment process may optionally connect one or more pressure ports of the cartridge and/or instrument. Alternatively, or in combination, the self-alignment process may engage one or more heat sources (e.g., thermal cycling heaters) of the instrument onto the cartridge.
  • the instrument comprises one or more actuators configured to interface with the cartridge.
  • the cartridge remains stationary within the instrument after insertion therein and one or more of the actuators can move in X, Y, and/or Z directions to interact with different elements of the cartridge as described herein, which may enable more than one element on the cartridge to be actuated by a single actuator.
  • both the cartridge and the actuators remain stationary in the XY plane and the number of actuators provided on the instrument corresponds to the number of elements to be actuated on the cartridge as described herein.
  • the instrument is configured to move the cartridge in X, Y, and/or Z therewithin, which may enable more than one element on the cartridge to be actuated by a single actuator.
  • Actuators may include, but are not limited to: heaters/chillers, bi-directional liquid and/or gas pumps, fluid valves, optical sensing modules of camera/illumination/lenses, barcode scanners, rare earth magnets, etc., as described herein.
  • the present disclosure provides a system for detecting target nucleic acids, the system including an instrument configured to interface with a cartridge.
  • the instrument comprises: (i) one or more pumps; (ii) at least one valve actuator; (iii) a light source configured to illuminate a detection region of the cartridge; and (iv) an optical sensor configured to detect one or more signals from the detection region;
  • the cartridge comprises: (i) a sample interface configured to receive a sample comprising one or more nucleic acids; (ii) one or more reagent capsules; (iii) a sample preparation region; (iv) a detection region; (iv) and a plurality of pump interfaces fluidically connected to the sample interface, the one or more reagent capsules, the sample preparation region, and/or the detection region via a plurality of valves;
  • the one or more pumps is configured to apply positive and/or negative pressure through the plurality of pump interfaces;
  • the at least one or more pumps is configured to apply positive and/or negative pressure through the pluralit
  • the instrument is no larger than 6” by 5” by 3.75”, though dimensions can be outside this range as desired (see FIGS. 1A-1B for exemplary dimensions).
  • part of the cartridge may be partially disposed outside of the instrument during testing. If necessary, an external accessory may be used to delineate loading and unloading areas for the cartridge.
  • the sample collection workflow is for the specimen to be collected with the patient and transported to an instrument within the facility but potentially in a separate room.
  • Other collection workflows may include the instrument being co-located with a patient.
  • the cartridge may be loaded into the instrument before a specimen is collected allowing for reagent incubations to begin.
  • a workflow may preclude reagent incubation while the specimen is being collected from the patient.
  • specimen container labeling of the patient/ specimen information for transfer into the patient record is performed via the instrument. Sample collection in the same location as the instrument could permit the patient/ specimen information to be entered directly into the instrument and the specimen inserted into the cartridge already in the instrument, thereby reducing or eliminating the information transfer step.
  • FIG. 42 depicts an exemplary sample collection workflow in which the sample is collected and provided to the cartridge prior to loading the cartridge into the instrument.
  • FIGS. 1A-1C shows an exemplary embodiment of an instrument along with two exemplary cartridge designs that might be used with the instrument. All dimensions are in inches.
  • the two-pump, 4.2-inch-long cartridge in FIG. 1A is capable of a reagent nucleic acid extraction with no washing steps. This extraction may be feasible for specimen matrixes such as from nasal swab collected specimens or vaginal swab collected specimens.
  • specimen matrixes such as from nasal swab collected specimens or vaginal swab collected specimens.
  • IB may incorporate multiple reagents for washing the inhibitors from the extracted nucleic acids and eluting the nucleic acids in a liquid reagent for the molecular detection reaction.
  • the instrument is designed to accept either cartridge, and other cartridges may be designed to fit the instrument which can carry out any variation of the methods described herein as desired, as will be understood by one of ordinary skill in the art.
  • a cartridge e.g., a two-pump, and/or 4.2-inch cartridge
  • a cartridge is used for tests for upper respiratory pathogens (such as SARS-CoV-2, influenza or respiratory syncytial virus), or for sexually transmitted diseases (such as those caused by Neisseria gonorrhoeae or Chlamydia trachomatis).
  • a cartridge e.g., a 3 -pump, and/or 6-inch cartridge
  • blood samples such as those for hepatitis or HIV
  • stool specimens such as those for norovirus or Salmonella or Clostridium difficile (toxin A/B).
  • the instrument of a system described herein comprises one or more of the following design elements described in more detail below: an instrument housing and chassis; a cartridge mover; a detector/ sensor system (e.g., optical module); a thermal control system (e.g., a thermal module); a valve actuator, a liquid reagent capsule actuator, one or more pumps, power; a controller (including embedded software); communications (including embedded software); device administration (software); and an assay engine (software).
  • a detector/ sensor system e.g., optical module
  • a thermal control system e.g., a thermal module
  • a valve actuator e.g., a liquid reagent capsule actuator, one or more pumps, power
  • a controller including embedded software
  • communications including embedded software
  • device administration software
  • an assay engine software
  • the cartridge of a system described herein comprises one or more of the following design elements: a cartridge housing; a sample receiver or sample chamber, an amplification/detection region (e.g., a DETECTR module); a liquid reagent capsule, a valve, a dried reagent chamber, a pneumatic pump interface (also referred to herein as an air displacement pump interface), and a waste chamber.
  • a cartridge housing e.g., a DETECTR module
  • a liquid reagent capsule e.g., a DETECTR module
  • a valve e.g., a valve
  • a dried reagent chamber e.g., a dried reagent chamber
  • a pneumatic pump interface also referred to herein as an air displacement pump interface
  • either or both of the instrument and cartridge comprise one or more of the following design elements: valve(s); fluidic drive(s); and reagent reservoir(s).
  • the valve(s) comprise rotary valve(s).
  • the fluidic drive(s) comprise pump module(s).
  • the reagent reservoir(s) comprises liquid reagent capsule(s) and/or dried reagent capsule(s).
  • the instrument/cartridge interface comprises mechanical alignment features for ensuring that design elements will properly align, connect, and/or mate when a cartridge is inserted into an instrument to process a molecular diagnostic test from a biological sample.
  • the instrument may be configured to operate a cartridge as shows in FIGS. 2B-2C for implementing the schematic workflow as shown in FIG. 2A.
  • nucleic acid extraction comprises a solid-phase capture step (e.g., the Boom method; see Example 1).
  • the instrument comprises 3 pumps, 3 heaters and 3 valve actuators.
  • the instrument may be limited to as few pumps, heaters, valve actuators and sensors as desired by the user.
  • the architecture may revolve around moving the cartridge to the actuators and sensors as shown in FIG. 14.
  • one optical sensor module, one thermal (e.g., heater) module, one pump module, and one rotary valve actuator may execute the assay with a moving cartridge.
  • the one pump module contains one air displacement pump and two pushers that move the reagent capsules down in the cartridge to open the seal in the cartridge, thereby exposing the liquid to the fluidics manifold and the action of the air displacement pump to move the liquids within the cartridge.
  • the instrument may receive information from a user and/or deliver information to the user via a graphical user interface (GUI).
  • GUI graphical user interface
  • the instrument may send and/or receive information via a network connection (e.g., via WiFi and/or Bluetooth connection(s)).
  • the cartridge and instrument perform functions together to process a nucleic acid test on a sample inserted into the cartridge.
  • the instrument will also receive information such as patient name and other demographic information such as age, gender, etc. from a user. This information can be entered via a touch screen display, a keypad, a separate mobile device, a laboratory information management (LIM) system connected to the instrument by a local area network connection (e.g., WiFi or Bluetooth), or the like, or any combination thereof.
  • the instrument will output information to the user.
  • the information may include test results and may be provided to the patient being tested and/or to a health care provider (HCP) if the HCP is involved in the testing.
  • HCP health care provider
  • the instrument will transmit information via a touch screen display and/or via a local area network connection such as WiFi or Bluetooth.
  • the instrument may be used in a clinic, lab, or office with HCP physically present during the assay. Altematively, or in combination, the instrument may interface remotely with one or more HCPs via the cloud (e.g., using a telehealth or remote health environment) and test results and patient information may be transmitted via a secure encrypted internet protocol.
  • the contacts may facilitate information transfer between the cartridge and the instrument.
  • the electronic contacts may facilitate transfer of information about the cartridge itself, including, but not limited to, date of manufacture, expected lifetime (or “use by” date), lot number, assay identification, and/or any relevant assay parameters (e.g., information about assay temperatures, sequence timings, etc.), or the like.
  • the contacts may carry digital, power, and/or analog signals between the instrument and the cartridge.
  • transfer of electronic information and/or power may be accomplished through near-field communication (NFC) and/or radio-frequency identification (RFID) electronic components.
  • NFC near-field communication
  • RFID radio-frequency identification
  • the instrument housing houses all the actuators and sensors, the power supply, the electronics control boards, and the input/output interface screen.
  • the sensors and actuators may be organized such that the heater system contacts the thinner side of the cartridge from the bottom and the optical module views the cartridge from the top.
  • the pump interface and reagent capsule actuators may contact a thicker side of the cartridge on the top and the rotary valve actuator may contact the cartridge at the bottom.
  • the cartridge mover may be oriented on a side of the cartridge.
  • a cartridge e.g., a 3 -pump, and/or 6-inch cartridge
  • has 3 working zones see, e.g., FIG. 1C, FIGS. 2B-2C, 8A-10A).
  • the cartridge e.g., a two-pump, and/or 4.2-inch cartridge
  • the cartridge has 2 working zones (see e.g., FIG. 1A, FIG. 7).
  • FIG. 15 shows the optical module’s field of view for 3 zones and a pump interface.
  • the reagent capsules can’t be seen; the liquid reagent capsules may be under the label and the label may be perforated and the circles separated from the label when the liquid reagent capsules are translated by the actuator.
  • FIG. 16 illustrates front (left) and back (right) views of the internal components of an exemplary instrument, highlighting a plurality of instrument modules that directly interface with a cartridge as it is moved through the exemplary instrument by a cartridge mover module.
  • the cartridge is not shown in FIG. 16. More details about the various modules are provided in greater detail below.
  • the internal instrument components comprise one or more of an optical module, a cartridge mover module, a pump module, a thermal/heater module, and a rotary valve module.
  • FIG. 17 shows the cartridge being inserted into the instrument. After insertion, the cartridge is moved into the open space between the four modules shown in FIG. 16.
  • FIG. 18 is an isometric front view of the instrument, showing the cartridge in the instrument with the instrument cover transparent.
  • FIG. 19 shows four of the instrument modules coupled to the cartridge. The rotary valve module is not shown as it is hidden by the cartridge.
  • the instrument comprises a cartridge mover.
  • the cartridge mover is a module within the instrument that interfaces with the cartridge to translate it to position the cartridge so as to interface with instrument modules that directly or indirectly contact the cartridge (e.g., the optical module, the heater module, the pump module, and the instrument actuator for the cartridge’s rotary valve).
  • the cartridge mover translates the cartridge linearly (e.g., bi-directionally in the x-y direction).
  • FIG. 20 is a CAD rendering of a cartridge mover module embodiment, illustrating how the instrument module contacts the cartridge when the cartridge is inserted into the instrument by the user as shown in FIG. 17.
  • Mechanical features e.g., teeth
  • gear teeth on the cartridge mover, in order to convert rotational motion of the cartridge mover gears into linear x-y motion of the cartridge.
  • the instrument comprises one or more fluidic drives.
  • the one or more fluidic drives may include one or more pump modules, one or more reagent capsule actuators, one or more rotary valve actuators, and/or one or more motorized XYZ gantries as described herein.
  • the number and composition of the fluidic drivers may be determined by the cartridge configuration and assay being run, as will be understood by one of ordinary skill in the art.
  • the instrument includes one or more pump modules.
  • the instrument comprises fewer pump drives than the number of pump interfaces on the cartridge.
  • the instrument may have a single pump that interfaces with each of two or more pump interfaces of the cartridge at different stages of operation.
  • the pump module(s) comprises an air displacement pump or other pneumatic system that applies positive and/or negative pressure to the pump interfaces to push and pull liquid bi-directionally within the cartridge.
  • one or more actuators may be provided in the pump module to translate reagent capsules down (Z-direction) onto a molded plastic piercer to pierce a seal on the respective reagent capsule and release the liquid therefrom into the fluid channels of the cartridge (see, e.g., FIGS. 25-27).
  • the liquid reagent capsules may be substantially similar to those described in PCT/US2022/034110, which is herein incorporated by reference in its entirety.
  • FIG. 25 An isometric bottom view of an instrument pump module is shown in FIG. 25.
  • FIG. 26 shows a view of the pump module in the instrument from a perspective within the cartridge housing.
  • the reagent capsule actuators also referred to herein as pushers
  • the air displacement pump interface can be seen through the cartridge housing to the right of the optical module and the cartridge mover.
  • FIG. 27 shows a side view of the pump module (top) and the rotary valve actuator (bottom) interfacing with a cartridge (middle, blurred for illustrative purposes).
  • the reagent capsule pushers are illustrated pushing down into the cartridge on two reagent capsules.
  • the reagent capsule pushers are independently controlled.
  • the reagent capsule pushers are used serially to dispense two reagents during an assay.
  • the reagent capsule pushers are used in parallel to dispense two reagents to be mixed together for an assay.
  • the rotary valve actuator comprises a motor driven actuator configured to contact a rotary valve on the cartridge when in use.
  • the rotary valve actuator may be moved away from the cartridge body when not in use (e.g., during cartridge movement). Rotating the actuator in either direction when in contact with the rotary valve rotates the rotary valve.
  • the actuator can be controlled by a stepper motor with a motor encoder.
  • the motor encoder ’s null or zero position is known by the encoder, and all rotated positions are known at all times.
  • a motorized XYZ gantry will be used to drive an actuator platform coupled to one or more moveable components of the cartridge (e.g., the sample reservoir, fluid jumpers, rotary valves, reagent reservoirs, syringes, etc.).
  • the X-direction motor will drive forward/backward movement
  • the Y-direction motor will drive left/right movement.
  • the Z motor may function as an actuator to drive a plunger up/down (e.g., to dispense from the reagents capsules as described herein).
  • an actuator will be used to drive a syringe capsule that generates positive and/or negative pressure to move fluids within the cartridge.
  • a pump in the instrument may be used to generate positive and/or negative pressure as described herein.
  • positive pressure can also be used to generate bubbles within a reservoir (e.g., to mix samples with lysing reagents and/or silica beads).
  • the instrument may comprise one or more sensors for monitoring pressure within the cartridge, which may be used to provide feedback and/or control a “kill switch” configured to stop operation should the pressure within the cartridge be outside of operating limits.
  • multiple locations within the cartridge might utilize one or more magnets of the instrument (e.g., to capture magnetic silica beads within a fluid reservoir, chamber, or channel as described herein). In some embodiments, these magnets may engage and disengage the cartridge via a motor or a solenoid. In some embodiments, an ultrasonic horn, waveguide, or probe may be used to agitate the sample and/or move fluids within the cartridge as described herein. In some embodiments, a single ultrasound source may be provided within the instrument and a multi-location waveguide may direct the ultrasound energy to various locations within the cartridge (e.g., the sample receiver interface, a sample reservoir, etc.).
  • the thermal environment of the cartridge may be controlled by the instrument.
  • the thermal system primarily has a controllable heating element and/or a controllable cooling element.
  • the thermal system may comprise a control loop configured for implementing direct and/or indirect temperature measurement of the fluid and/or heating or cooling the fluid to reach a pre-determined temperature.
  • the control loop can include one or more feedback element to facilitate temperature control.
  • the control loop can include one or more model-based control elements to facilitate temperatures control.
  • the thermal system may be configured to perform thermocycling and/or maintain a constant temperature within one or more regions or zones of the cartridge as described herein.
  • At least one thermal (e.g., heater) module may be implemented.
  • only one thermal module may be used.
  • a thermal controller implements a Peltier-based system in order to facilitate quick changes in temperature.
  • a lysis temperature might be 65°C
  • an elution temperature might be 80°C
  • the DETECTR reaction temperature may be 58°C.
  • the components of an illustrative thermal module are listed in the Table 3 and illustrated in FIG. 22, with an exploded diagram in FIG. 23.
  • FIG. 24 shows the thermal module in a cutaway view of the instrument. The cartridge mover is to the left of the heater module and the rotary valve actuator to the right in this illustration.
  • the instrument uses Peltier/thermoelectric (TEC) coolers to heat and/or cool one or more regions or zones of the cartridge.
  • the TEC coolers may engage with the cartridge upon insertion, thereby achieving intimate contact with the cartridge at the desired region(s) or zone(s).
  • the cartridge may be configured to provide improved thermal conductivity and/or time response for use with the various heating/cooling systems described herein.
  • a surface of the cartridge may comprise a thin film for heat exchange with the heater/cooler(s) of the instrument.
  • the cartridge may be configured to enable temperature measurement(s).
  • the instrument may use one or more thermal reservoirs to heat and/or cool one or more regions or zones of the cartridge.
  • the thermal reservoir(s) may be moved in and out of contact with a surface of the cartridge in order to conduct heat therebetween.
  • one or more thermal reservoirs may be moved in and out of contact with the surface in order to heat and cool the fluid in a cyclic manner (e.g., for thermocycling-based amplification).
  • one or more thermal reservoirs may be moved in and out of contact with the surface in order to maintain a desired temperature of the fluid for a pre-determined length of time (e.g., for isothermal amplification).
  • one or more thermal reservoirs may be moved into contact with a surface of the cartridge to generate one or more heat zones and the fluid may be moved into and out of the heat zones (and optionally between heat zones when multiple thermal reservoirs are provided) in order to heat and/or cool the fluid in a cyclic manner and/or for a pre-determined length of time.
  • the thermal reservoir(s) may have a higher thermal capacity that the fluid(s) disposed within the one or more regions or zones of the cartridge, thereby enabling rapid heat transfer from the thermal reservoir(s) to the fluid(s).
  • moving the thermal reservoir(s) into and out of contact with the cartridge may provide a simpler alternative to moving the fluid (e.g., via pumping or by moving the cartridge itself) between different temperature zones to change the temperature of the fluid.
  • the instrument uses optical or photonic heating.
  • the instrument directly heats the reaction mixture by irradiating the liquid with optical wavelengths that are strongly absorbed by water (e.g., infrared wavelengths targeting water absorption peaks below 1500nm) or by transducing light to heat using particles (e.g., gold nanoparticles) dispersed throughout the heated region(s) or zone(s) of the cartridge (e.g., using light of additional wavelengths).
  • direct heating may offer advantages in thermal speed compared to some indirect heating methods by directly heating the reaction mixture instead of controlling the temperature of a surface in contact with the reaction mixture.
  • direct heating may reduce or avoid time delays associated with fluids inside a chamber equilibrating with a surface temperature.
  • optical temperature measurement may be performed within the heated region(s) or zone(s), e.g., by a spot measurement or a camera.
  • the optical temperature measurement device measures the temperature by quantifying infrared radiation emitted by the fluid and the chamber enclosing the fluid.
  • optical temperature measurement may be advantageous in terms of speed, as measurement loops involving such devices may have the potential to be faster than some other temperature measurement devices. This may allow faster control loop response times and therefore faster transitions between temperatures within a region or zone.
  • the cartridge may comprise one or more resistive heating elements and the instrument may be configured to apply an electric current to the one or more resistive heating elements to heat the fluid.
  • the resistive heating element(s) may be disposed adjacent a surface of one or more regions or zones of the cartridge to be heated.
  • the electrical contacts of the instrument may engage with one or more contact pads of the resistive heating elements of the cartridge upon insertion, thereby achieving electrical communication with the cartridge in order to heat the desired region(s) or zone(s).
  • resistive heating may provide a simple and flexible alternative or addition to other heating mechanisms which may require more complex instrumentation, a larger instrument and/or cartridge footprint/form factor, and/or more complicated fluidic designs.
  • the instrument may use one or more fans to facilitate cooling of the fluid within the cartridge (e.g., using forced convection).
  • the fan(s) may be used in combination with any of the heating mechanisms described herein to control the temperature of the fluid within the cartridge.
  • temperature control may be important for controlling one or more reactions taking place on the cartridge.
  • temperature control may be important for controlling a lysis reaction, an amplification reaction(s) in the amplification region, and/or a detection reaction in the detection region.
  • temperature control of +/- 0.5°C may be desired for some assays steps, such as during PCR annealing (which may be conducted at a temperature within a range from about 45.0°C to about 70.0°C).
  • other assay steps may have less stringent temperature control designs.
  • the temperature control system may be sufficient with a temperature accuracy of +/- 2.0°C.
  • the precision for temperature control within the system may be to a tenth of a degree Celsius.
  • the instrument may comprise a detector/sensor system to read out the spatial multiplexing implemented in the cartridge.
  • the detector system may comprise a source and a detector.
  • the source may comprise an illumination source.
  • the source may comprise an electrical signal source.
  • the detector comprises an optical sensor or optical detector.
  • the detector is an image sensor (e.g., a camera, photomultiplier tube, charge- coupled device, active-pixel sensor, photodiode, complementary metal-oxide semiconductor (CMOS), or the like).
  • the detector may comprise an array of discrete optical detectors, one for each detection spot or chamber within the detection region of the cartridge.
  • the detector may comprise one or more optical channels. For example, a first optical channel may be used to detect cleavage of a reporter and a second optical channel may be used to detect background signal (e.g., autofluorescence).
  • one or more lenses and/or filters may be implemented to create an image with sufficient resolution and brightness for detection of reporter cleavage.
  • one or more digital masks may be used to process an optical signal detected by the detector (e.g., a digital mask may be used to remove bubbles from the analysis).
  • the detector performs multiple jobs.
  • a first job may be the detection of fluorescence signal from a detection reaction (e.g., a DETECTR reaction) in the detection region/module of the cartridge.
  • a second job may be to image labels for recordkeeping and image barcodes for the instrument’s software to decipher the encoded information.
  • the optical module design has two illumination capabilities - one each for the described two jobs. In some embodiments, the two different illuminators are not used in parallel. For imaging labels or barcodes, the visible/red LED may be turned on. For imaging a fluorescence signal from the DETECTR module, fluorescence excitation LED may be turned on.
  • the imaging system enables fluorescence detection to be performed on all of the microwells in the detection region at the same time, as opposed to a single point reader which would require the microwells to be read one at a time (in sequence).
  • the imaging system does not require any moving parts to read all of the wells in the cartridge. As a result, this type of reader may generally be manufactured at lower cost in comparison to a single point reader and its associated scanning mechanics.
  • FIG. 21 shows an exemplary layout of the instrument’s fluorescence imaging system.
  • the main components include the camera assembly (including camera sensor chip, lens and filter), fluorescence excitation LED (and corresponding filter) and the visible/red LED and photodiode.
  • the relative position of these components in cross section is shown below FIG. 21 along with the position of the cartridge.
  • the resolution of the detector may be about 50 micrometers. In some embodiments, the resolution of the detector may be less than about 50 micrometers. In some embodiments, the resolution of the detector may be less than about 25 micrometers. In some embodiments, the resolution of the detector may be less than about 5 micrometers.
  • the signal is selected from the group consisting of an optical signal, a fluorescent signal, a colorimetric signal, a potentiometric signal, an amperometric signal, and a piezo-electric signal.
  • the signal is associated with a change in an index of refraction of a solid or gel volume in which said at least one programmable nuclease probe is disposed. Cleavage of a reporter (e.g., a protein-nucleic acid) can produce a signal.
  • the signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample.
  • cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal.
  • Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample.
  • the sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
  • optical sensors e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies
  • SPR surface plasmon resonance
  • interferometric sensors or any other type of sensor suitable for detecting calorimetric signals, potenti
  • the method for detection is fluorescence.
  • the detector (or plurality of detectors) may detect a change in wavelength (e.g., a change in color), intensity (e.g., brightness), or a degree of wavelength change (e.g., with appropriate dispersion elements to access wavelength space).
  • the system may be configured to detect one or more wavelengths (e.g., one for each fluorophore in a fluorescence- based system).
  • optical filtering of illumination light and/or detection light may be implemented to block unwanted crosstalk (e.g., illumination light being detected in detection systems).
  • one or more optical elements may be implemented in the cartridge and/or camera to aid in image acquisition and control. Such elements may include alignment features, fiduciary marks, and/or controls for detector level-setting (e.g., high, low, and zero response spots or chambers within the device). In some embodiments, the one or more optical elements may facilitate image registration, level calibration, and/or error detection on an automated basis.
  • detection mechanisms may comprise interferometry, surface plasmon resonance, electrochemical detection such as potentiometry, or other detection mechanisms.
  • power for the instrument may be provided via a voltage autoswitching from 100 VAC to 240 VAC electrical mains connection with a rechargeable lithium- ion battery that has sufficient energy storage to complete one or more assays. Recharging of the battery can be automatically managed by the instrument when connected to a mains electrical outlet.
  • a controller module houses the embedded computer, ROM and RAM memory for the embedded computer and dedicated integrated circuits to control all of the instrument’s actuators and sensors.
  • the instrument has Bluetooth and/or WiFi capability for electronic data connections to nearby computers.
  • communications are via one or more of a user touch screen, a USB connector, and an ethernet connector.
  • FIG. 31 and FIG. 32 show the controller and communications module of an exemplary instrument.
  • the embedded software that manages actuator movement and data from the optical module and the other sensors that control actuators is maintained on the instrument’s controller. Additional modules of software may be included in the instrument.
  • a device administration manages the cybersecurity and data transfer into and out of the instrument.
  • another module of software called an assay engine, stores the assay protocols to execute each assay for the specific cartridge inserted into the instrument. The labeling on the cartridge, as scanned by the optical module, may inform the assay engine which assay protocol to use after interpretation by the embedded software.
  • a cartridge for the analysis and detection of one or more nucleic acids in a sample may be coupled to an instrument in order to analyze the one or more nucleic acids as described herein.
  • the cartridge may be configured to receive a sample and perform one or more reactions on the sample in order to detect one or more target nucleic acids.
  • the cartridge may be configured to perform one or more of the following steps: sample collection, sample extraction, sample lysis, protein degradation, nucleic acid extraction, nucleic acid purification, nucleic acid concentration, waste removal, nucleic acid elution, nucleic acid amplification, a programmable nuclease-based detection reaction, target detection, and/or reporter detection, or any combination thereof.
  • FIGS. 8A-10A, 10C show how a longer cartridge (e.g., the 3-pump cartridge of FIG. 1C and 2B-2C) can be organized and designed to interface with the instrument and to provide the functionality illustrated in the schematics described in FIGS. 2A-6.
  • the design of a shorter, simpler, cartridge e.g., the two-pump cartridge of FIG. 1C
  • Additional information related to the rotary valve(s), dried reagent chamber(s), and detection module are provided below.
  • one or more elements or components of the cartridge may be modular (or have a modular design). Any of the cartridges described herein may comprise one or more modules. The one or more modules may be independently created, modified, replaced, and/or exchanged with other modules or between different systems. The one or more modules may be selected or replaced as desired in order for the cartridge to perform a desired function and/or assay.
  • the cartridge may comprise a plurality of different modules. In some embodiments, the cartridge may comprise one or more of a sample receiver module, a reagent module, a sample preparation/concentration module, an amplification module, a mixing module, a detection module, or any combination thereof.
  • the cartridge may comprise a sample receiver module, a reagent module, a sample preparation module, an amplification module, and a detection module.
  • the cartridge may comprise more than one of the same type of module (e.g., two or more reagent modules, etc.).
  • two or more of the modules may be in fluid communication with each other.
  • two or more of the modules may be capable of being put in fluid communication with each other (e.g., via a valve such as a rotary valve, jumper valve, or the like).
  • the one or more modules may be physically distinct from one another.
  • the one or more modules may be functionally distinct from one another.
  • the one or more modules may be manufactured individually or as a unitary construction (in which the modules are functionally distinct but physically part of a same physical construct).
  • the cartridge may comprise one or more pieces or segments configured to be coupled to one another to form a complete cartridge.
  • providing the cartridge in more than one piece may facilitate proper reagent storage.
  • some of the reagents needed for the assay may be in liquid form while other reagents may be in dried, vitrified, or lyophilized form.
  • Preferred storage conditions for liquid form reagents may be sufficiently different from preferred storage conditions for dried, vitrified, or lyophilized reagents so as to make it difficult or impossible to store each reagent in its preferred condition. Separating the liquid reagents from the dried, vitrified, and/or lyophilized reagents onto different cartridge segments may enable proper reagent storage for each segment.
  • the cartridge in accordance with some embodiments provides one or more of: a reagent reservoir, a sample interface in fluid communication with the reagent reservoir, the sample interface configured to receive a sample, an amplification region in fluid communication with one or more of the sample interface or the reagent reservoir and configured to amplify one or more nucleic acids in the sample, and a detection region in fluid communication with the amplification region.
  • a reagent reservoir in fluid communication with the reagent reservoir
  • the sample interface configured to receive a sample
  • an amplification region in fluid communication with one or more of the sample interface or the reagent reservoir and configured to amplify one or more nucleic acids in the sample
  • a detection region in fluid communication with the amplification region.
  • Each of the reagent reservoir, the sample interface, the amplification region and the detection region may be provided as one or more separate modules, as described herein.
  • one or more reagent reservoir may comprise a reagent capsule as described herein.
  • the reagent capsule may be disposed within a reagent silo as described herein.
  • each liquid reagent may be individually filled and sealed in a reagent capsule.
  • the reagent capsule may be filled with a dry reagent.
  • the number of reagent reservoirs (e.g., reagent capsules) disposed on a reagent module may be tailored to the specific reagents required by the assay being performed by the system.
  • the number of reagent reservoirs in some embodiments may vary from 4 to 6 in number. In some embodiments, there may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 reagent reservoirs per module.
  • the cartridge may comprise a sample receiver configured for sample preparation.
  • the cartridge may comprise one or more reagent reservoirs comprising one or more lysis reagents.
  • the cartridge may comprise one or more waste reservoirs in fluid communication with the sample receiver.
  • the cartridge may comprise a one or more reagent reservoirs comprising one or more wash buffers and/or elution buffers.
  • the one or more reagent reservoirs and the waste reservoir may be connected to the sample receiver by one or more fluid channels and valves.
  • fluid flow within the cartridge may be unidirectional.
  • a sample collector may be inserted into the sample interface as described herein.
  • the sample interface may comprise a sample reservoir.
  • the sample interface may optionally comprise a scraper as described herein.
  • Lysis reagents may be transferred from one or more reagent reservoirs to the sample reservoir for lysis of the sample as described herein.
  • lysis of the sample may completely kill any live pathogens within the sample.
  • additional chemical inactivation reagents may be transferred from the reagent reservoir(s) into the sample reservoir in order to ensure complete inactivation (e.g., killing) of the sample pathogen.
  • the sample reservoir or a downstream location therefrom may be heated to a predetermined temperature for a predetermined amount of time in order to ensure complete sample inactivation.
  • one or more additional reagents e.g., PK for protein lysis, alcohol for nucleic acid aggregation, etc.
  • PK for protein lysis
  • alcohol for nucleic acid aggregation
  • nucleic acids of the sample may be captured by a concentrator (e.g., a filter, column, beads, mesh, etc.) within or downstream of the lysis zone (e.g., within a jumper or fluid channel connecting the lysis zone to an amplification/detection module).
  • the sample may be transferred to a separate sample concentration region comprising a concentrator downstream of the lysis zone. Wash and/or elution reagents may be transferred from one or more reagent reservoir to the concentrator for washing and elution of the sample as described herein. Elution of the nucleic acids from the concentrator may move the purified nucleic acids downstream into an optional amplification module or detection module.
  • the amplification module may comprise one or more amplification channels or chambers as described herein.
  • the nucleic acids may be amplified as described herein and then transferred downstream to a detection module. In some embodiments, the nucleic acids may be amplified in the detection module prior to or during detection. In some embodiments, the nucleic acids may not be amplified. One or more target nucleic acids may then be detected in a multiplexed fashion as described herein.
  • various modules of a cartridge are fluidically connected to one another by a plurality of valves.
  • valving is provided to accomplish the fluid schematics described FIGS. 2A-7.
  • a rotary valve with seven ports is provided.
  • two cartridge rotary valves are utilized for a simple extraction method in a shorter cartridge (see, e.g., Example 2).
  • three cartridge rotary valves are used for a cartridge implementing a nucleic acid capture and elution extraction method (e.g., a Boom method; see Example 1).
  • all seven ports are utilized for Valve B.
  • valve B utilizes an inlet to outlet path for all the fluid motion states (that is, there are no positions in which fluid does not flow through the valve).
  • the cartridge rotary valve comprises one or more, e.g., four, components (see, e.g., the exploded diagram of FIG. 11).
  • the illustrated components may be plastic.
  • Part A includes the fluid holes that act as the valve inlet and outlet ports.
  • Part B is an elastomeric sealing gasket that prevents leaking.
  • Part B can be affixed to Part C by over-molding adhesion of the Part B and Part C plastics.
  • Part D compresses the combined Parts B and C to Part A.
  • Part D can be joined to Part A by laser welding or ultrasonic welding or the like.
  • the joining of Part D to Part A also forms fluid paths on the cartridge bottom.
  • a similar joining of another plastic part to the top of Part A also provides fluidic chambers and channels but these are not shown clearly in FIG. 11.
  • FIG. 31 shows the rotary valve actuator contacting a transparent and blurred cartridge for illustrative purposes.
  • three rotary valves on the exemplary cartridge are in a position that allows the Part C portion of the rotary valve (shown in FIG. 11) to slide between the prongs of the cartridge and the rotary valve actuator. Rotating the actuator in either direction then contacts Part C and rotates the rotary valve.
  • the actuator can be controlled by a stepper motor with a motor encoder. In some embodiments, the motor encoder’s null or zero position is known by the encoder, and all rotated positions are known at all times.
  • FIG. 30 shows the bottom of the cartridge with the three rotary valves in positions for loading and unloading the cartridge.
  • the cartridge comprises a plurality of liquid reagent capsules.
  • the liquid reagent capsules described herein may be substantially similar to those described in PCT/US2022/034110, which is herein incorporated by reference in its entirety.
  • the number of capsules within the cartridge may vary depending on the number and volume of reagents.
  • a module may comprise a plurality of capsules varying from 4 to 8 in number. In some embodiments, there may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules per module.
  • the fluid volume in each capsule may vary from about 50 pL to 1 mL per capsule. In some embodiments, the fluid volume in each capsule may be at least about 100 pL, about 200 pL, about 300 pL, about 400 pL, about 500 pL, about 600 pL, about 700 pL, about 800 pL, about 900 pL, or about 1000 pL.
  • the fluid volume filled in a capsule may vary from about 50 pL to 500 pL. In some embodiments, the fluid volume in a capsule may be at least about 50 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, or about or 500 pL.
  • one or more reagent capsules may be filled with more liquid than is needed for its intended purpose. In some embodiments, only a portion of this volume may be used for an assay and excess residual volume may be left in the reagent capsule (i.e., the reagent capsule may be configured to dispense a fraction of the liquid disposed therein) and/or routed to a waste reservoir on the cartridge or instrument.
  • the capsule(s) may be fully assembled onto the cartridge during manufacturing. In some embodiments, the capsule(s) may be provided with the cartridge in a modular fashion so as to allow a user to mix and match reagents as desired.
  • the instrument may be configured to open each capsule inside the cartridge at a predetermined time at which the liquid inside the capsule is needed.
  • the instrument may provide fluid movement from the capsule by translating the capsule within the silo from a closed configuration to an open configured, thereby displacing the liquid therefrom into the space within the silo as described herein.
  • moving the capsule within the silo to pierce the pierceable cover may open a vent and improve subsequent fluid movement from the capsule.
  • translation of the reagent capsule(s) may be actuated by an actuator on the instrument (e.g., by a capsule actuator on a pump module of the instrument).
  • an actuator on the instrument e.g., by a capsule actuator on a pump module of the instrument.
  • one or more reagent capsule may be positioned at a defined location such that the instrument’s actuator may be shared by stepper motors or solenoids for the movement.
  • the cartridge comprises a plurality of dried reagent chambers.
  • the cartridge comprises locations for up to five dried reagent chambers.
  • These reagent chambers can hold lyophilized (e.g., freeze-dried) reagents or simply dried reagents.
  • the dried reagent chambers may be filled separately from the rest of the cartridge, in order to allow for insertion into the drying oven or the lyophilizer during cartridge manufacture and assembly.
  • the chambers with the dried reagents are then attached to the cartridge (e.g., via laser welding of plastic materials). Ultrasonic welding or heat staking are alternative methods for connecting the dried reagent chambers to the cartridge.
  • the types of reagents that can be in the dried reagent chambers include, but are not limited to, lysis agents, protein digestion enzymes such as Proteinase K, activator salts for the DETECTR reaction and polymerase and CRISPR enzymes for the DETECTR reaction.
  • the oligonucleotides that are specific for each test’s DETECTR reaction may be dried or lyophilized in wells/chambers of the detection region.
  • FIG. 12 shows an exemplary embodiment of the cartridge with five already-attached (fully-assembled) dried reagent chambers. Illustrations of an exemplary dried reagent chamber, and how it may be pierced for assembly are provided in FIG. 10B.
  • the body is injected molded, then a top film is either heat staked or welded to form the top of the capsule prior adding the dried reagents.
  • a bottom film may then be added to the bottom of the capsule body to keep the reagents sterile prior to fully assembling onto the cartridge body. Piercers on the cartridge body pierce the bottom film upon assembly and put the dried reagents in fluid communication with the fluid channels of the cartridge.
  • a valve may open up a fluid pathway to/from the dried reagent chamber, and liquid can be passed through one of the piercers, into the dried reagent capsule body where it meets and rehydrates the dried reagents, and then out through the other piercer. If needed, the liquid can be passed back and forth through the capsule in order to ensure full rehydration and recapture of the dried reagents.
  • the cartridge may comprise a sample concentrator. Any of the cartridges described herein may comprise any of the sample concentrators described herein.
  • the sample concentrator may be downstream of the sample interface and may comprise one or more structures configured to capture, purify, and/or concentrate nucleic acids of the sample.
  • the sample concentrator may be upstream of the amplification region.
  • the sample concentrator may be in a sample concentration module.
  • the sample concentrator may be in a reagent module.
  • the sample concentration may be in the sample reservoir, a jumper, or a concentrator reservoir.
  • the sample concentrator may be in fluid communication with one or more reagent reservoirs comprising one or more nucleic acid concentration reagents.
  • the concentrator may comprise one or more of a filter, a membrane, a column, a mesh, a surface, or one or more beads configured to capture nucleic acids dispose within or downstream of the sample reservoir and configured to capture, purify, and/or concentrate nucleic acids from the sample.
  • the sample concentrator may be configured to facilitate liquidliquid extraction, solid-liquid extraction, and/or solid-phase extraction techniques.
  • solid-phase extraction may be preferred for smaller sample volumes and/or improved cartridge-based performance (e.g., by reducing solvent volumes and/or simpler workflow automation).
  • Solid-phase nucleic acid purification may utilize a silica-based method such as the Boom method.
  • the concentrator may comprise one or more silica particles (e.g., silica beads or silica magnetic beads), glass fiber filters, silica-coated membranes/meshes/filters, or the like.
  • Application of chaotropic salts such as guanidinium thiocyanate or guanidinium hydrochloride may facilitate adsorption of the nucleic acids onto the silica-based concentrator.
  • Alcohol may be used to wash away salts and cellular debris that may contaminate or inhibit downstream reactions such as amplification.
  • the purified nucleic acids may then be eluted off the concentrator (e.g., using a moderate salt buffer).
  • solid-phase nucleic acid purification may utilize charge switching for nucleic acid concentration.
  • the concentrator may comprise one or more particles (e.g., beads or magnetic beads) or surfaces (e.g., membranes, meshes, filters, etc.) functionalized with an ionizable material such as chitosan.
  • the ionizable material may, for example, be provided as a coating on the one or more particles or surfaces.
  • the ionizable material may be pH-responsive and allow nucleic acid capture in moderately low pH (in which the ionizable material is cationic) and nucleic acid release in moderately high pH.
  • the purified nucleic acids may be eluted off the concentrator with a buffer compatible with downstream reactions such as amplification.
  • charge switching may provide a less complicated workflow and/or reduce or eliminate the use of inhibiting reagents compared to some other purification methods.
  • the cartridge as described herein may comprise a sample concentration module.
  • the sample concentration module provides some similar functions of the sample receiver module.
  • the concentration module may comprise a concentration reservoir comprising one or more of a filter, a column, a membrane, a mesh, a surface, or one or more beads configured to capture nucleic acids.
  • the concentrator may comprise one or more of a filter, a column, a membrane, a mesh, a surface, or one or more beads configured to capture nucleic acids.
  • the beads may be silica beads.
  • the beads may be silica-coated beads.
  • the beads may be silica-coated magnetic beads.
  • the beads may be chitosan-coated beads.
  • the beads may be chitosan-coated magnetic beads.
  • the filter may be a glass fiber filter.
  • the membrane may be a chitosan-functionalized membrane (e.g., a nylon membrane coated with chitosan).
  • the concentrator may comprise more than one type of material configured to capture nucleic acids.
  • the concentrator may comprise one or more silica bead and one or more chitosan-coated beads.
  • the concentrator may comprise one or more magnetic beads. In some embodiments, the concentrator may comprise one or more silica-coated magnetic beads.
  • the sample concentration module includes a liquid reservoir configured to interface with a magnet on the instrument to immobilize the magnetic beads to a surface of the cartridge. In some embodiments, the magnet may be moved away from the cartridge by the instrument when the beads are to be released downstream to the amplification module. In some embodiments, the cartridge may contain a filter mesh downstream of the magnet to capture the magnetic beads. In some embodiments, a liquid back flow may move the magnetic beads functionally downstream to the amplification module. In embodiments, the magnetic beads may remain immobilized throughout the sample concentration process and the sample nucleic acids may be eluted therefrom and flown downstream while the magnetic beads remain immobilized in the concentration region.
  • sample concentration may include one or more washing steps (e.g., to remove unwanted proteins and/or salts from the nucleic acids bound to the concentrator).
  • concentrated nucleic acids with 260/280 and 260/230 ratios may be released by the concentrator by an elution buffer as described herein.
  • the sample concentrator may retain bound nucleic acids to beads in a filter mesh or with a magnet as described herein in order to allow for a thorough elimination of unwanted salts and proteins that might interfere with downstream amplification and/or detection reactions.
  • the beads may be magnetized.
  • the beads may not be magnetized.
  • the last step of the concentration may include a release of the nucleic acids from the magnetic beads or filter.
  • the elution liquid may be a low to no salt reagent configured to release the nucleic acids from the concentrator surfaces.
  • any of the cartridges described herein may comprise one or more reagents for concentration of nucleic acids.
  • one or more of the concentration reagents may be liquid reagents.
  • liquid reagents for concentration of nucleic acids may purify and/or concentrate the nucleic acids with a high degree of purity.
  • the liquid reagents may include a series of buffers having different pH.
  • the liquid reagents may include a series of wash reagents of different ionic strength with alcohols of different concentrations.
  • high purities of nucleic acids may be obtained by gradually changing the ionic strength and alcohol concentration of the reagents until the final reagent, known as the elution buffer, facilitates the release of the purified nucleic acids from the concentrator.
  • the purity of the released nucleic acids may be measured spectrophotometrically at 260 nanometers.
  • impurities may be measured at 230 nanometers and 280 nanometers.
  • ratios of signal at 260/280 should be 1.8 for DNA and 2.0 for RNA. Lower 260/280 ratios may indicate a low capture of nucleic acid or a high amount of protein in the eluate.
  • ratios of signal at 260/230 should be within a range of about 2.0 to 2.2.
  • a low signal at 260/230 could mean a high amount of salt such as guanidine in the elution.
  • unwanted salts or proteins can inhibit the downstream amplification and contamination reactions.
  • the reagents for concentration may be housed in a different set of reagent capsules (e.g., on a different reagent module) than the initial sample preparation reagents (e.g., lysis reagents, etc.) as described herein.
  • the number and volume of reagent capsules used for nucleic acid concentration may be different (e.g., fewer in number) and/or of a larger or smaller volume than those used for sample collection and lysis.
  • the number of capsules for a concentration module may vary from 4 to 8 in number. In some embodiments, there may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 capsules.
  • the fluid volume in each capsule may vary from 100 pL to 1 mL per capsule. In some embodiments, the fluid volume in each capsule may be at least about 100 pL, about 200 pL, about 300 pL, about 400 pL, about 500 pL, about 600 pL, about 700 pL, about 800 pL, about 900 pL, or about 1000 pL.
  • the cartridge may comprise a filter, mesh, magnet, etc. configured to prevent downstream travel of the concentrator s).
  • the concentrator may comprise the filter or mesh.
  • the concentrator may comprise a magnetic particle capable of being immobilized with a magnet of the instrument.
  • the concentrator may comprise a particle sized and shaped for capture by the filter or mesh.
  • the cartridge may comprise an amplification region. Any of the cartridges described herein may comprise any amplification region described herein.
  • the amplification region may be downstream of the sample reservoir, reagent reservoir(s), and/or concentrator. In some embodiments, the amplification region may upstream of the detection region. In some embodiments, the amplification region may overlap with the detection region (e.g., for assays in which amplification and detection occur in the sample location).
  • the amplification region may be in an amplification module. In some embodiments, the amplification region may be in an amplification/detection module.
  • the amplification region may comprise one or more amplification chambers or channels.
  • the amplification region may comprise one or more amplification reagents.
  • one or more amplification reagents may be stored in one or more reagent reservoirs and mixed with the sample prior to, during, or after transfer of the sample to the amplification region.
  • the system may be configured to amplify one or more target nucleic acids in the amplification region using the one or more amplification reagents.
  • one or more target nucleic acids may be specifically reproduced in the amplification via an isothermal reaction, such as LAMP, or a thermocycling reaction, such as polymerase chain reaction (PCR), as described herein.
  • the amplification reaction may include reverse transcription (RT), for example when amplifying an RNA target nucleic acid.
  • the amplification region may comprise two or more independently controllable temperature zones corresponding to two or more separate channels or chambers therein.
  • the amplification region may include at least three separate channels or chambers with independent temperature control. In at least some instances, independent temperature control may facilitate the performance of different amplification reactions in each of the three zones.
  • one or more amplification reagent may be deposited in the amplification channel or chamber as a dried, vitrified, or lyophilized reagent.
  • Introduction of the liquid sample e.g., nucleic acid eluate from the sample reservoir or sample preparation module
  • the reagents for amplification may hydrate the dried, vitrified, or lyophilized reagent(s).
  • one or more amplification reagents may be dried, vitrified, or lyophilized in situ (e.g., after deposition in the amplification channel or chamber or in a jumper upstream of the amplification channel or chamber).
  • the amplification reagents may include one or more amplification enzymes, oligonucleotide primers, nucleotides, etc. as described herein.
  • the reagents may include a small amount of liquid configured to provide a specific amount of activator salts (e.g., metal ions) in liquid form to facilitate the amplification reaction.
  • the amount of activator salts may vary from 10 pL to 25 pL.
  • the liquid may be pre-aliquot into three separate volumes for each of the three amplification channels (in embodiments comprising three amplification channels, for example) in the amplification region.
  • the sample liquid and the amplification reagents may be mixed in the amplification region and/or in the microfluidic channel between the reagent reservoir(s) and the amplification channel or chamber.
  • the cartridge may comprise a detection region. Any of the cartridges described herein may comprise any detection region described herein.
  • the detection region may be downstream of an amplification region.
  • the detection region may be co-localized with the amplification region (e.g., when amplification and detection occur in the same location and/or at the same time).
  • the detection region may be in a detection module.
  • the detection region may be in an amplification/detection module.
  • the detection region may comprise one or more detection locations. In some embodiments, a detection location may comprise one or more detection reagents.
  • the detection location may comprise a reporter, a guide nucleic acid, and/or a programmable nuclease.
  • the reporter may be any of the reporters, or any combination of reporters, described herein.
  • the guide nucleic acid may be any of the guide nucleic acids, or any combination of guide nucleic acids, described herein.
  • the programmable nuclease may be any of the programmable nucleases, or any combination of programmable nucleases, described herein. Any one of several programmable nucleases (e.g., Cas proteins) may be used individually or in combination with other programmable nucleases.
  • the programmable nuclease may comprise a Casl2, Casl3, Casl4, or CasPhi family Cas protein.
  • the programmable nucleases may be different in the different detection locations.
  • FIG. 39 depicts a surface 1301 comprising an immobilized programmable nuclease- guide nucleic acid complex (1302, 1303) and a plurality of reporters 1304, with one reporter having been cleaved by an activated programmable nuclease 1302.
  • one or more detection reagents may be immobilized on a surface 1301 of the detection region.
  • one or more programmable nuclease 1302, one or more guide nucleic acid 1303, and/or one or more reporter 1304 may be immobilized on a surface 1301 of the detection region.
  • one or more detection reagents may be immobilized on a surface 1301 of the detection region via a linker 1305.
  • the programmable nuclease 1302 may be complexed with a guide nucleic acid 1303 complementary to a specific target nucleic acid sequence as described herein.
  • the programmable nuclease 1302 may be activated by binding of the guide nucleic acid 1303 (which is complexed thereto) to the target nucleic acid. Activation of the programmable nuclease 1302 may enable trans-cleavage of the reporter 1304 as described herein.
  • the reporter 1304 may comprise a detection moiety 1306 as described herein.
  • the reporter 1304 may comprise a detection moiety 1306 (e.g., a fluorophore) and a quencher moiety 1307 (e.g., a quencher) configured to generate a detectable signal when separated from one another by release of a cleaved portion 1308.
  • a detection moiety 1306 e.g., a fluorophore
  • a quencher moiety 1307 e.g., a quencher
  • trans-cleavage of the reporter 1304 by the activated programmable nuclease 1302 may release the detection moiety 1306 (or a quencher moiety 1307, depending on the signal), thereby generating a signal indicative of the presence or absence of the target nucleic acid in the sample as described herein.
  • the reporter 1304 may comprise any of the reporters 1304 described herein, and may comprise any detection moiety 1306 described herein (and/or other moieties or molecules described herein which facilitate signal detection). As shown in FIG. 39, cleavage of the reporter 1304 may release the quencher
  • cleavage of the reporter 1304 may result in a non-fluorescent signal as described herein.
  • cleavage of the reporter 1304 can produce a calorimetric signal, a potentiometric signal, an amperometric signal, a colorimetric signal, or a piezo-electric signal.
  • the one or more detection reagents can be immobilized in discrete detection locations using NHS-amine chemistry as described herein.
  • a primary amine-modified guide nucleic acid and a primary amine-modified reporter may be conjugated to an NHS-coated surface of the detection region.
  • the one or more detection reagents may be immobilized using streptavidin-biotin chemistry as described herein.
  • a biotinylated reporter and a biotinylated guide nucleic acid may be immobilized to a streptavidin-coated surface of the detection region.
  • the one or more detection reagents may be immobilized using maleimide-thiol chemistry as described herein.
  • a thiol-modified guide nucleic acid and a thiol-modified reporter may be conjugated to a maleimide-coated surface of the detection region.
  • the one or more detection reagents may be immobilized using epoxy-amine chemistry as described herein.
  • an amine-modified guide nucleic acid and an amine-modified reporter may be conjugated to an epoxy-coated surface of the detection region.
  • the one or more detection reagents may be immobilized using hydrogels as described herein.
  • an acrydite-modified guide nucleic acid and an acrydite-modified reporter may be co-polymerized with an acrylate-modified oligomer (e.g., PEG-diacrylate) prior to deposition on the surface 1301 of the detection region or in situ on the surface 1301 of the detection region.
  • an acrylate-modified oligomer e.g., PEG-diacrylate
  • one or more detection reagent may be immobilized to a surface (e.g., surface 1301 shown in FIG. 39) of the detection location as described herein.
  • one or more detection reagents may be dried, vitrified, or lyophilized and deposited on a surface of the detection location.
  • deposition of the dried, vitrified, or lyophilized may be accomplished using photolithography, inkjet printing, or other patterning techniques.
  • one or more detection reagents may be dried, vitrified, or lyophilized in situ (e.g., after deposition on a surface of the detection location).
  • the reagents may be spotted in a microwell array (e.g., as shown in FIGS. 13, 33A-38D).
  • Specific wells may contain reagents for specific target nucleic acids.
  • the one or more detection reagents e.g., the guide nucleic acid and reporter
  • the detection locations may be patterned as an array.
  • an array of detection locations may comprise a two-dimensional array of detection spots, chambers, or microwells arranged in orthogonal directions.
  • the array may be an m x n array having m columns of n detection spots, chambers, or microwells arranged in rows.
  • m and n may be different.
  • m and n may be the same.
  • an array of detection locations may be asymmetrical (e.g., detection locations may be patterned to minimize the usage of space in the detection region 1401 with regard for symmetry). It will be apparent to one of ordinary skill in the art that the detection region 1401 may comprise any suitable number of detection locations and the detection locations may be arranged in any suitable manner so as to enable multiplexed target nucleic acid analysis.
  • the detection reagents may be provided on a surface of the detection region as an immobilized array.
  • the array may comprise a number of detection locations within a range of about 1 to about 200, within a range of about 3 to about 200, or within a range of about 10 to about 200.
  • the array may comprise at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
  • one or more locations may comprise the same detection reagents (e.g., detection reagents specific to the same target nucleic acid).
  • 1 to 12 detection locations may comprise the same detection reagents.
  • an exemplary detection region may comprise anywhere from 1 to 12 replicate spots for each target nucleic acid to be detected.
  • the detection region may comprise an array of detection spots.
  • Each detection spot of the array may comprise a reporter and a different programmable nuclease of a plurality of programmable nucleases as described herein.
  • each of the different programmable nucleases of the plurality of programmable nucleases may comprise a different guide nucleic acid which is complementary to a different target nucleic acid of a plurality of target nucleic acids.
  • the reporter and each different programmable nuclease and/or guide nucleic acid of each detection spot of the array may be immobilized to a surface of the detection region.
  • each different programmable nuclease may be configured to cleave an adjacent reporter and generate a different signal of a plurality of signals. Each different signal may therefore be indicative of the presence or absence of a different target nucleic acid.
  • the reporter may comprise a fluorophore and a quencher as described herein.
  • the guide nucleic acid and reporter may each be immobilized to a surface of a detection spot with a linker as described herein.
  • the target nucleic acids may be freely available within the fluid volume of the detection region.
  • multiple guide nucleic acids for a single target nucleic acid may be combined within a single detection spot in order to increase a rate of reaction. Localizing the guide nucleic acids and reporter may localize the detectable signal for each target nucleic acid to the detection spot, thus enabling the spatial multiplexing.
  • the cartridge comprises a detection region.
  • the detection region comprises a plurality of chambers that are fluidically connected to one another.
  • An exemplary layout of a detection region (e.g., DETECTR module) of some cartridge embodiments is show in FIG. 13.
  • the wells may be contained within an area of - 32mm x 32mm.
  • Each well may be designed to be less than or equal to about lOpL in volume.
  • the wells may be nominally 1.15mm deep and ⁇ 4mm in length. Additional embodiments of reaction wells/chambers in a microfluidic device compatible with cartridges herein are described in PCT/US2022/034110, which incorporated herein by reference.
  • the wells are filled with fluid sequentially via an entrance port (shown on the bottom of FIG. 13). Air may be exhausted through an exit channel, as seen in the central area with a membrane attached to the rear of the cartridge.
  • the detection region may comprise a plurality of chambers (e.g., microwells 3901 shown in FIGS. 33A-38D).
  • each chamber may comprise a reporter and a different programmable nuclease of a plurality of programmable nucleases.
  • each of the different programmable nucleases of the plurality of programmable nucleases may comprise a different guide nucleic acid which is complementary to a different target nucleic acid of a plurality of target nucleic acids.
  • one or more programmable nuclease, one or more reporter, and/or one or more guide nucleic acid may be lyophilized, vitrified, or dried as described herein.
  • each different programmable nuclease may be configured to cleave an adjacent reporter and generate a different signal of a plurality of signals. Each different signal may therefore be indicative of the presence or absence of a different target nucleic acid as described herein.
  • the detection region may comprise a plurality of chambers.
  • Each of the plurality of chambers may have a volume within a range of about 0.5 pL to about 10 pL.
  • each of the plurality of chambers may have a volume of about 0.75 pL, about 1 pL, about 1.5 pL, about 3 pL, about 5 pL, or about 10 pL.
  • the volume of each chamber may be within a range of about 0.5 pL to about 10 pL while maintaining an aspect ratio sufficient to enable filling of the chambers while still preventing cross-talk between chambers as described herein.
  • the plurality of chambers may comprise a plurality detection channels, chambers, microwells, nanowells, or the like.
  • the plurality of chambers may comprise a plurality of microwells disposed downstream of an amplification chamber (or downstream of nucleic acid purification if amplification is not performed or is performed in the detection microwells themselves).
  • the plurality of microwells may be arranged and filled in series or in parallel with one another following nucleic acid amplification.
  • the amplification channel may be connected to the plurality of chambers by one or more loading channels.
  • each loading channel may lead to a single detection chamber.
  • each loading channel may lead to a plurality of detection chambers.
  • the plurality of detection chambers may begin filling sequentially. In some embodiments, the plurality of detection chambers may be filled simultaneously.
  • One or more capillary valves may be arranged at the inlet to each detection chamber (e.g., microwell 3901 shown in FIGS. 33A- 33B).
  • the capillary valves may facilitate controlled loading of detection chambers and/or minimize cross-talk between detection chambers.
  • a loading channel e.g., loading channel 3902 shown in FIGS. 33A-33B
  • a capillary valve may be positioned at an inlet to each of the plurality of detection chambers.
  • the capillary valve inlet area may be much smaller than that of the main loading channel.
  • the fluid typically at least partially (e.g., 25-50%) fills a first detection chamber before the fluid has enough pressure build up to start going into the capillary valve.
  • the fluid may then continue down the path provided by the loading channel from one detection chamber/capillary valve to the next.
  • the fluid volume input to the detection region is such that the volume is equal to the amount of volume in each detection chamber, air clears the plenum behind each detection chamber. If pressure is held, the fluid in each detection chamber cannot come out of the detection chamber and interfere with the reactions occurring in a separate detection chamber.
  • a surface of the detection region may comprise an air- permeable/liquid-impermeable membrane such as a hydrophobic membrane or a plurality of hydrophobic membranes (e.g., membrane 4012 shown in FIG. 34B).
  • the surface may be a top or bottom surface of the detection region.
  • the surface may interface with a heater or be opposite a surface which interfaces with the heater.
  • the air-permeable/liquid-impermeable membrane may be configured enable gas to pass therethrough for venting but reduce or prevent liquid from exiting the detection region.
  • each detection chamber may have a surface (e.g., a bottom surface) comprising air-permeable/liquid- impermeable membrane (e.g., a hydrophobic membrane).
  • the air-permeable/liquid-impermeable membrane may comprise any material which enables gas but not liquid to vent therethrough.
  • a hydrophobic membrane may comprise a woven polypropylene or polyethylene membrane having an average pore size of about 0.20 microns. The burst pressure of the membrane may be selected based on the loading pressures used to fill the detection region in order to facilitate loading without substantial liquid loss through the membrane.
  • a sample liquid may include a surfactant or other additive that reduces surface tension (e.g., TWEEN).
  • a surfactant or other additive that reduces surface tension
  • membranes that are more hydrophobic and/or oleophobic may be preferred in order to avoid wetting.
  • the membrane comprises polytetrafluoroethylene (PTFE).
  • the air-permeable/liquid-impermeable membrane may comprise the surface of the detection region configured to interface with a heater. Without being bound by any particular theory, it is believed that after loading with the reaction liquid the wetted membrane may bring thermal transfer advantages as the liquid causing it to swell may improve thermal transfer between the heater and the reaction liquid during isothermal reactions by improving temperature accuracy, time to reach a desired temperature, and/or temperature stability.
  • FIGS. 33A-33B show illustrative representations of microfluidic devices comprising a plurality of chambers 2303 fluidically connected in sequence, in accordance with some embodiments.
  • Each of the illustrated chambers 2303 comprises a well 3901, an inlet channel 3902, an outlet 3903 (also referred to as an outlet port), and a capillary valve 3904 (for simplicity, only some of each of these structures is labeled in the figures).
  • the locations of wells 3901, capillary valves 3904, and outlets 3903 are labeled.
  • FIGS. 33A and 33B present alternative arrangements of the capillary valves 3904 and outlets 3903 relative to the respective wells 3901.
  • the fluid movement is laminar, due to surface tension.
  • the inlet channel 3902 is 0.40 mm by 0.35 mm in width 3906 and depth 3907, respectively.
  • the capillary valves 3904 are oriented such that they branch off of the main channel 3902 at an angle of about 90° or greater.
  • the capillary valve 3904 dimensions are 0.30 mm by 0.20 mm in width and depth, respectively.
  • the capillary valve 3904 inlet area is much smaller than that of the inlet channel 3902 between the wells 3901.
  • the fluid fills a well 3901 of an upstream chamber by about 10% to about 100% (e.g. about 25-50%) before there is adequate pressure resulting in the fluid migrating into the capillary valve 3904 and into the next chamber 2303 downstream in the sequence.
  • the fluid then continues filling the well 3901 of the upstream chamber while the fluid also progresses down the path from the upstream well 3901 and capillary valve 3904 to the next. In this way, fluid enters each chamber sequentially, but at any given time, one or more upstream chambers may continue to fill in parallel with one or more downstream chambers.
  • the wells 3901 have a volume of approximately 5 pL to 15 pL (e.g., about 10 pL).
  • maintained pressure in the wellplate prohibits fluid from coming out of the well containing the fluid, initially.
  • the outlet 3903 is connected to the well 3901 by an outlet channel 3905.
  • FIG. 33B shows several chambers 2303 wherein the plenum 3902 is located directly above each well 3901.
  • the inlet channel 3902 of a given chamber intersects the well 3901 at one point along the well perimeter and the capillary valve 3904 intersects at a separate point along the well perimeter (e.g., opposite one another across the well).
  • an outlet 3903 corresponds to an opening of the respective well 3901, which may optionally be sealed by an air-permeable membrane (not shown).
  • the back of the microfluidic device is covered with a hydrophobic membrane allowing the passage of gases, including air.
  • the hydrophobic membrane does not allow the transport of fluids at standard operating pressures.
  • fluid movement results from surface tension.
  • the main channel 3902 is 0.50 mm by 0.35 mm in width and depth, respectively.
  • each well 3901 is roughly 0.50 mm in diameter.
  • the capillary valves 3904 are oriented such that they are directly over each well 3901.
  • the capillary valve 3904 dimensions are 0.30 mm by 0.20 mm in width and depth, respectively.
  • the capillary valve inlet area is much smaller than that of the main channel 3902.
  • fluid flowing in the microfluidic device substantially fills the well 3901 before there is adequate pressure in the fluid for the fluid to begin migration into the capillary valve. The fluid then continues down the path from one well 3901 and capillary valve to the next well in sequence.
  • FIGS. 34A-34C depict different views of a microfluidic device in accordance with some embodiments.
  • FIG. 34A shows a perspective view of a top surface of a detection region 1401 of the microfluidic device.
  • FIG. 34B shows a perspective cross-sectional view of the top surface of the detection region 1401.
  • FIG. 34C shows a plan view of the top surface of the detection region 1401.
  • the illustrated microfluidic device comprises a plurality of chambers 2303 fluidically connected in sequence, in which each chamber of the plurality of chambers comprises a well 3901, an inlet channel 3902, an outlet 3903, and a capillary valve 3904.
  • the arrangement of features in the device and fluid flow within the device is similar to those illustrated in FIG. 33A.
  • the chambers branch off of a common plenum, with the opening of the inlet channel 3902 to each chamber (except the first chamber in the sequence) defined by a capillary valve 3904 having a smaller cross-sectional area relative to the plenum at the intersection thereof.
  • the chambers are optionally arranged in a circular pattern in the device.
  • the outlets 3903 are arranged closer to the center of the circular pattern to save space of the device, and terminate in openings at the bottom of the device that are sealed with an air-permeable, liquid-impermeable membrane 4012 (indicated in the figure as a hydrophobic membrane attached with adhesive, heat staked, laser welded, or ultrasonic welded).
  • the base of the device may be infrared absorbent and/or opaque (4006), which may be beneficial for reducing noise when using light-based detection methods.
  • the device may include a top lid that is infrared transmissive and/or clear (4007) (e.g., to facilitate detection of reactions in the wells 3901 when using light-based detection methods).
  • the device may include a barb fitting inlet 4010 for loading of sample fluid.
  • FIG. 34B illustrates the optional location 4011 of one or more detection reagents, which may be dried down or in lyophilized form within the well 3901 of the chamber prior to the introduction of a sample fluid, as described herein.
  • FIG. 34C further illustrates a laser weld path 4013 around the channels, chambers, and outlets of the detection region 1401.
  • FIGS. 34A-34C also illustrates that exits extending from the wells 3901 to their respective outlets 3903 may be of alternating long (4004) and short (4005) lengths, rather than all having a single uniform length, in order to maximize the use of space and minimize the overall size footprint of the detection region, which may be beneficial for manufacturing, user adoption, and/or interaction between the device and an instrument (e.g., a heater, detector, etc.).
  • FIG. 35 depicts a perspective view of a bottom surface of the detection region 1401 of the microfluidic device, including optional features 4014 around the outlets to help ultrasonically weld an air-permeable membrane to the device.
  • FIG. 36 depicts a perspective view of a top surface of an embodiment of the microfluidic device in which a film is used to form a top surface of the chambers instead of a molded lid.
  • FIG. 37 depicts a perspective cross-sectional view of an embodiment of the microfluidic device in which the membrane can be compounded with an adhesive backing to adhere to the device instead of using laser welding or ultrasonic welding or heat staking.
  • the adhesive later 4301 can be cut with the same hole pattern as the chip and adhered to the membrane.
  • the membrane does not allow the transport of fluids therethrough at standard operating pressures.
  • a sample liquid may include a surfactant or other additive that reduces surface tension (e.g., TWEEN).
  • a surfactant or other additive that reduces surface tension
  • membranes that are more hydrophobic and/or oleophobic may be preferred in order to avoid wetting.
  • the membrane comprises polytetrafluoroethylene (PTFE).
  • PTFE polytetrafluoroethylene
  • FIGS. 38A-38D depict different views of an exemplary detection region 1401 of a microfluidic device in accordance with some embodiments.
  • FIGS. 38A-38B shows perspective and plan views of the top of the device.
  • FIGS. 38C-38D shows perspective and plan views of the bottom of the device.
  • the illustrated device includes a plurality of chambers 2303 fluidically connected in sequence, and connected to an upstream first inlet channel 4401 by way of a bubble purge channel 4403 (or a bubble trap) disposed therebetween.
  • bubbles in the liquid of the first inlet channel 4401 enter the bubble purge channel 4403 and escape through a membrane 4404 that is hydrophobic and/or oleophobic and which covers the bottom surface of the bubble purge channel 4403.
  • the liquid itself is substantially prevented from passing through the membrane and instead continues to flow along the bubble purge channel 4403 towards its exit into a second inlet channel 4402, reduced in or substantially free of bubbles.
  • the second inlet channel 4402 feeds into the plurality of chambers 2303. Also illustrated is the location of the hydrophobic and/or oleophobic membrane 4404 (which may be the same as or different from membrane 4012 shown in FIG. 34B) that covers at least a portion of a bottom surface of the device.
  • Non-limiting examples of membrane materials include polypropylene, polyethylene, PTFE, PTFE with polypropylene backing, polycarbonate, PVC, and PVDF.
  • the arrangement of features in the device and fluid flow within the device is similar to those illustrated in FIG. 33A.
  • the chambers branch off of a common plenum, with the opening of the inlet channel to each chamber (except the first chamber in the sequence) defined by a capillary valve 3904 having a smaller cross-sectional area relative to the plenum at the intersection thereof.
  • the chambers are arranged in a circular pattern in the device.
  • the outlets 3903 are arranged closer to the center of the circular pattern, and terminate in openings at the bottom of the device that are sealed with the membrane.
  • the base of the device may be infrared absorbent and opaque 4006.
  • the device may include a top lid or membrane that is infrared transmissive and clear 4007 (e.g., to facilitate detection of reactions in the wells 3901).
  • the hydrophobic or oleophobic membrane 4404 does not allow the transport of fluids at standard operating pressures. In some embodiments, fluid movement results from surface tension.
  • the ratio of well width to well depth is less than 1.
  • the chambers or the wells thereof comprise a volume of approximately 0.75 pL.
  • the fluid volume input to the microfluidic device is such that the volume is equal to the amount of volume in each chamber or the wells thereof, which results in air clearing the plenum behind each well.
  • the fluid in each well is substantially confined to the well, thereby limiting interference with reactions occurring in separate fluidically connected chambers.
  • each chamber of the plurality of chambers comprises a well, an inlet channel, an outlet, and a capillary valve; the capillary valve of each chamber (i) has a cross-sectional area that is smaller than a cross-sectional area of the inlet channel of the respective chamber, and (ii) forms an entrance of the inlet channel of the next chamber in the sequence; and each outlet is air- permeable and configured to retain liquid within the respective chamber.
  • each chamber further comprises detection reagents comprising a guide nucleic acid and a reporter.
  • each guide nucleic acid comprises a targeting sequence that hybridizes with a target nucleic acid of a plurality of different target nucleic acids or an amplicon thereof, and (ii) is effective to form a complex with a programmable nuclease that is activated upon binding the corresponding target nucleic acid or amplicon thereof;
  • the guide nucleic acid of a first chamber in the plurality of chambers comprises a different targeting sequence from the guide nucleic acid of a second chamber in the plurality of chambers;
  • each reporter comprises a cleavable nucleic acid and a detection moiety, and (ii) is configured to be cleaved to form a detectable cleavage product in response to activation of the complex in the well of the respective chamber.
  • Non-limiting examples of guide nucleic acids, programmable nucleases, reporters, and detection moieties are described herein, including with respect to various other aspects and embodiments.
  • cleavage of the reporter in response to activation of the complex upon binding the corresponding target nucleic acid forms a detectable product.
  • the nature of the detectable product and how it is detected may vary depending on the nature of the detection moiety.
  • the reporter may comprise a fluorescent label and a quencher, with cleavage of the reporter releasing the quencher and permitting detection of the fluorescent label upon excitation at the appropriate wavelength.
  • the reporter may comprise an enzyme (e.g., horseradish peroxidase) that is either in an inactive form or is physically separated from its substrate, with cleavage of the reporter releasing the enzyme to act upon its substrate, and the enzyme activity detected (e.g., as in a color change).
  • an enzyme e.g., horseradish peroxidase
  • the capillary valve has a cross-sectional area that is about 75%, 50%, 25%, 15%, or less than a cross-sectional area of the inlet channel of the respective chamber. In some embodiments, the capillary valve has a cross-sectional area that is about 50% or less than a cross sectional area of the inlet channel. In some embodiments, the respective cross-sectional areas of the capillary valve and the inlet are the cross-sectional areas of each at a point where the capillary valve and inlet intersect. In some embodiments, the respective cross- sectional areas of the capillary valve and the inlet are the cross-sectional areas of each at the point where it intersects the well.
  • the capillary valve is oriented at an angle of about 90° or greater (e.g., about 100°, 120°, 140°, 160°, or 180°) with respect to the inlet channel of the respective chamber.
  • the capillary valve forms a junction with the inlet channel of the respective chamber (see, e.g., FIG. 33 A).
  • the capillary valve and the inlet channel intersect the well of a respective chamber at separate points along a perimeter of the well (see, e.g., FIG. 33B).
  • the inlet channels comprise a width of about 0.3 mm to about 0.6 mm (e.g., about 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm, 0.55 mm, or 0.6 mm) and a depth of about 0.25 mm to about 0.45 mm (e.g., about 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, or 0.45 mm).
  • the inlet channels comprise a width of about 0.4 mm and a depth of about 0.35 mm.
  • the inlet channels comprise a width of about 0.5 mm and a depth of about 0.35 mm.
  • the capillary valves comprise (a) a width of about 0.2 mm to about 0.4 mm (e.g., about 0.2 mm, 0.25 mm, 0.3 mm, or 0.4 mm) and a depth of about 0.1 mm to about 0.3 mm (e.g., about 0.1 mm, 0.15 mm, 0.2 mm, 0.25 mm, or 0.3 mm). In some embodiments, the capillary valves comprise a width of about 0.3 mm and a depth of about 0.2 mm.
  • each of the wells has an internal volume of about 0.1 pL to about 50 pL, 0.5 pL to about 20 pL, or about 1 pL to about 10 pL. In some embodiments, each of the wells has an internal volume of about 0.75 pL. In some embodiments, each of the wells has an internal volume of about 10 pL.
  • the outlet comprises an opening sized to permit displacement of air therethrough but to retain liquid within the well under an operating pressure of the microfluidic device.
  • the opening will be smaller than the capillary valve, such that at the operating pressure, fluid is directed through the capillary valve, rather than the outlet (through which, air is still allowed to pass).
  • the outlet comprises a surface comprising a hydrophobic coating. In some embodiments, the outlet comprises a surface comprising an air-permeable membrane that is hydrophobic and/or oleophobic. In some embodiments the outlet functions as a vent. In some embodiments, the outlet is open to air. In some embodiments, the outlet is connected to a pneumatic system. In some embodiments, each outlet is connected to a pressure source, which may be the same or different. In some embodiments, each outlet in the system is connected to a common pressure source. In some embodiments, the pneumatic system provides positive pressure to the microfluidic system. In some embodiments, the pneumatic system provides negative pressure to the microfluidic system.
  • the outlet comprises a surface comprising a hydrophobic membrane
  • any of a variety of suitable hydrophobic membranes may be used.
  • the hydrophobic membrane comprises a woven polymer (e.g., woven polypropylene or woven polyethylene).
  • the hydrophobic membrane comprises pores of about 0.1 microns to about 3 microns in size (e.g., about 0.1 microns, 0.25 microns, 0.5 microns, 0.75 microns, 1 micron, 1.5 microns, 2 microns, or 2.5 microns).
  • the hydrophobic membrane forms a bottom surface of the respective well.
  • the hydrophobic membrane comprises a woven polymer with pores of about 0.1 microns to about 2 microns in size, and forms a bottom surface of the respective well.
  • the outlet comprises a surface comprising a oleophobic membrane
  • any of a variety of suitable oleophobic membranes may be used.
  • the oleophobic membrane comprises a woven polymer.
  • the oleophobic membrane comprises pores of about 0.1 microns to about 3 microns in size (e.g., about 0.1 microns, 0.25 microns, 0.5 microns, 0.75 microns, 1 micron, 1.5 microns, 2 microns, or 2.5 microns).
  • the oleophobic membrane forms a bottom surface of the respective well.
  • the oleophobic membrane comprises a woven polymer with pores of about 0.1 microns to about 2 microns in size, and forms a bottom surface of the respective well.
  • the system further comprises a sample interface configured to receive a sample, wherein the sample interface is in fluid communication with the plurality of chambers. Examples of sample interfaces are described herein, such as in connection with various other embodiments.
  • the sample interface is fluidically connected to the plurality of chambers via one or more sample preparation regions.
  • the one or more sample preparation regions comprise a lysis region or zone configured to lyse one or more components of the sample.
  • the lysis region comprises lysis reagents.
  • Non-limiting examples of lysis reagents include proteases (e.g., proteinase K), chaotropic agents, detergents, salts, and solutions of high osmolality, ionic strength, and pH.
  • the one or more sample preparation regions comprise an amplification region.
  • the amplification region comprises amplification reagents. Examples of amplification reagents are described herein, such as in connection with various other embodiments.
  • the system comprises one or more heaters for use in controlling the temperature of one or more regions of the microfluidic device (e.g., a lysis region, an amplification region, and/or the plurality of chambers).
  • heaters are disclosed herein, including heating elements that can also function as cooling elements (e.g., Peltier/thermoelectric coolers).
  • the heater is in thermal communication with a surface of the microfluidic device.
  • thermal communication is effected by integration of a heater within the microfluidic device.
  • thermal communication is effected by physical contact between the heater and the microfluidic device.
  • thermal communication is effected by placing the heater in proximity to a surface of the microfluidic device, without direct contact therewith. In some embodiments, thermal communication is effected by placing the heater in contact with a thermal conductor (e.g., a heat spreader) in direct contact with a surface of the microfluidic device.
  • the heater is moveable within the system, and can be brought into thermal contact (and optionally direct contact) with the microfluidic device when needed, then separated from the microfluidic device when no longer needed.
  • the outlets vent through a first surface of the microfluidic device, and the heater is in thermal communication with a second surface of the microfluidic device, where the first surface is opposite the second surface.
  • the microfluidic device may comprise any suitable number of chambers. In some embodiments, all of the chambers in the microfluidic device are fluidically connected. In some embodiments, a microfluidic device comprises two or more sets of chambers, in which chambers within a set are fluidically connected in sequence, but chambers in different sets may or may not be connected. For example, two or more sets of chambers may each branch off from a common sample interface or sample preparation region. Alternatively, two or more sets of chambers in a single microfluidic device may be unconnected from each other and fed by separate sample interfaces. In some embodiments, the plurality of chambers comprises at least 10, 25, 50, 75, 100, 150, 200, 300, 400, 500, 1000 chambers fluidically connected in sequence. In some embodiments, the plurality of chambers comprises at least 10, 25, 50, or 100 chambers fluidically connected in sequence. In some embodiments, the plurality of chambers comprises at least 50 chambers fluidically connected in sequence.
  • the detection reagents further comprise a programmable nuclease.
  • a programmable nuclease A variety of suitable programmable nucleases are available. Non-limiting examples of programmable nucleases are provided herein, such as in connection with various other embodiments.
  • the programmable nuclease comprises a Cas protein. Non-limiting examples of Cas proteins include Cas 12, Cast 3, Cas 14, CasPhi, and thermostable Cas proteins.
  • the detection reagents further comprise amplification reagents. Examples of amplification reagents are disclosed herein, such as in connection with various other embodiments.
  • Amplification reagents may include one or more primers and a polymerase (e.g., a DNA polymerase).
  • the detection reagents are in a lyophilized form.
  • the guide nucleic acid and/or the reporter in each chamber are immobilized, dried, or otherwise deposited to a surface of the respective chamber.
  • immobilization is by a linkage.
  • the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, an interaction (e.g., a covalent or noncovalent bond) between members of a binding pair (e.g., streptavidin and biotin), an amide bond, or any combination thereof.
  • Non-limiting examples of linkages for immobilizing reagents to a surface are described herein, such as in connection with various other embodiments.
  • the present disclosure provides microfluidic devices comprising a loading channel and a plurality of chambers.
  • the loading channel comprises a first capillary valve disposed upstream of a second capillary valve disposed therein.
  • the plurality of chambers comprises a first chamber fluidically coupled to the loading channel upstream of the first capillary valve.
  • the microfluidic device comprises a second chamber fluidically coupled to the loading channel between the first capillary valve and the second capillary valve.
  • the microfluidic device comprises a third chamber fluidically coupled to the loading channel downstream of the second capillary valve.
  • each chamber of the first, second, and third chambers comprises an outlet.
  • each of the first and second capillary valves have a cross-sectional area that is smaller than a cross-sectional area of the loading channel.
  • each outlet is gas-permeable and configured to retain liquid within the respective chamber.
  • the loading channel, plurality of chambers, and capillary valves may be sized in accordance with various embodiments described herein.
  • the chambers may comprise one or more reagents, such as one or more detection reagents, in accordance with various embodiments described herein. V. CERTAIN METHODS OF USE
  • any of the systems described herein may be used to detect one or more target nucleic acids in a sample.
  • detecting the one or more target nucleic acids may comprise one or more of the following steps: sample collection, sample extraction, sample lysis, protein degradation, nucleic acid extraction, nucleic acid purification, nucleic acid concentration, waste removal, nucleic acid elution, nucleic acid amplification, a programmable nuclease-based detection reaction, target detection, and/or reporter detection, or any combination thereof.
  • the present disclosure provides methods for detecting one or more of a plurality of different target nucleic acids in a system described herein.
  • the method comprises, (a) flowing a liquid comprising one or more of the different target nucleic acids or amplicons thereof into the plurality of chambers; (b) in one or more of the wells, forming the activated complex and cleaving the reporters; and (c) detecting the detectable cleavage products in one or more of the wells, wherein the location of a well comprising a detectable cleavage product identifies the target nucleic acid or amplicon thereof present in the well.
  • a given well at a particular location will comprise one or more guide nucleic acids of known sequence that hybridizes with a particular target nucleic acid.
  • formation of a detectable cleavage product in the given well is indicative of the presence of the corresponding target nucleic acid for that guide nucleic acid associated with the particular well at the known location.
  • detectable cleavage products may form in multiple wells, identifying the presence of each of the different target nucleic acids in the sample.
  • a detectable product may not form in one or more, most, or even all chambers, indicating that the corresponding target nucleic acids of the respective guide nucleic acids are not present above a threshold for detection.
  • methods of the present disclosure provide detecting the presence, absence, or amount of a plurality of different target nucleic acids in a sample.
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
  • the terms “upwardly”, “downwardly”, “vertical”, “horizontal”, and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
  • first and second may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/ element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent, depending on the context.
  • a “subject” can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject may be diagnosed with or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed with or suspected of being at high risk for the disease.
  • zzz vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample is an “zzz vitro" assay.
  • zzz vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • In vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), or +/- 10% of the stated value (or range of values).
  • Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.
  • treatment and “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • thermostable and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.
  • percent identity refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment.
  • an amino acid sequence is X% identical to SEQ ID NO: Y refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y in an alignment between the two.
  • computer programs may be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci.
  • a “one-pot” reaction refers to a reaction in which more than one reaction occurs in a single volume alongside a programmable nuclease-based detection (e.g., DETECTR) assay.
  • a programmable nuclease-based detection e.g., DETECTR
  • sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof and programmable nuclease-based detection (e.g., DETECTR) assays are carried out in a single volume.
  • amplification and detection are carried out within a same volume or region of a device (e.g., within a detection region).
  • Readout of the detection (e.g., DETECTR) assay may occur in the single volume or in a second volume.
  • the product of the one-pot DETECTR reaction e.g., a cleaved detection moiety comprising an enzyme
  • another volume e.g., a volume comprising an enzyme substrate
  • HotPot refers to a one-pot reaction in which both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reactions occur simultaneously.
  • a HotPot reaction may utilize a thermostable programmable nuclease which exhibits trans cleavage at elevated temperatures (e.g., greater than 37C).
  • amplification and “amplifying,” as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.
  • nucleic acid refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T/U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid.
  • the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand.
  • the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end
  • the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end.
  • Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.
  • cleavage assay refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid.
  • the cleavage activity may be cis- cleavage activity.
  • the cleavage activity may be trans-cleavage activity.
  • Assays which leverage the transcollateral cleavage properties of programmable nuclease enzymes are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions.
  • DETECTR DNA endonuclease targeted CRISPR trans reporter
  • detection of reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as “DETECTR”.
  • detecttable signal refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.
  • detecting a nucleic acid and its grammatical equivalents, as used herein refers to detecting the presence or absence of the target nucleic acid in a sample that potentially contains the nucleic acid being detected.
  • effector protein refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid.
  • the complex comprises multiple effector proteins.
  • the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid.
  • the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid.
  • effector protein refers to a protein that is capable of modifying a nucleic acid molecule (e.g., by cleavage, deamination, recombination). Modifying the nucleic acid may modulate the expression of the nucleic acid molecule (e.g., increasing or decreasing the expression of a nucleic acid molecule).
  • the effector protein may be a Cas protein (i.e., an effector protein of a CRISPR-Cas system).
  • guide nucleic acid refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein.
  • the first sequence may be referred to herein as a spacer sequence.
  • the second sequence may be referred to herein as a repeat sequence.
  • the first sequence is located 5’ of the second nucleotide sequence.
  • the first sequence is located 3’ of the second nucleotide sequence.
  • nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and/or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid.
  • a modification e.g., chemical modification, nucleotide sequence, or amino acid sequence
  • compositions or systems described herein refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system.
  • a composition may include an effector protein and a guide nucleic acid that do not naturally occur together.
  • an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by human intervention.
  • PAM protospacer adjacent motif
  • reporter and “reporter nucleic acid,” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.
  • sample generally refers to something comprising a target nucleic acid.
  • the sample is a biological sample, such as a biological fluid or tissue sample.
  • the sample is an environmental sample.
  • the sample may be a biological sample or environmental sample that is modified or manipulated.
  • samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.
  • target nucleic acid refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein.
  • a target nucleic acid may comprise RNA, DNA, or a combination thereof.
  • a target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).
  • target sequence refers to a sequence of nucleotides that hybridizes to a portion (preferably an equal length portion) of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.
  • Embodiment 1 A system for detecting target nucleic acids, the system comprising an instrument configured to interface with a cartridge; wherein:
  • the instrument comprises: (i) one or more pumps; (ii) at least one valve actuator; (iii) a light source configured to illuminate a detection region of the cartridge; and (iv) an optical sensor configured to detect one or more signals from the detection region;
  • the cartridge comprises: (i) a sample interface configured to receive a sample comprising one or more nucleic acids; (ii) one or more reagent capsules; (iii) a sample preparation region; (iv) a detection region; (iv) and a plurality of pump interfaces fluidically connected to the sample interface, the one or more reagent capsules, the sample preparation region, and the detection region via a plurality of valves;
  • the one or more pumps is configured to apply positive and/or negative pressure through the plurality of pump interfaces;
  • the at least one valve actuator is configured to actuate the plurality of valves
  • one or more pumps and at least one valve actuator are operable to move fluid between the sample interface, the one or more reagent capsules, the sample preparation region, and the detection region;
  • the detection region comprises a plurality of chambers that are fluidically connected to one another.
  • Embodiment 2 The system of Embodiment 1, wherein each chamber of the plurality of chambers further comprises detection reagents comprising a guide nucleic acid and a reporter, and further wherein:
  • each guide nucleic acid comprises a targeting sequence that hybridizes with a target nucleic acid of a plurality of different target nucleic acids or an amplicon thereof, and (ii) is effective to form a complex with a programmable nuclease that is activated upon binding the corresponding target nucleic acid or amplicon thereof;
  • the guide nucleic acid of a first chamber in the plurality of chambers comprises a different targeting sequence from the guide nucleic acid of a second chamber in the plurality of chambers;
  • each reporter comprises a cleavable nucleic acid and a detection moiety, and (ii) is configured to be cleaved to form a detectable cleavage product in response to activation of the complex in the respective chamber.
  • Embodiment 3 The system of Embodiment 1 or 2, wherein (a) the one or more reagent capsules comprises dried reagents; (b) the cartridge comprises one or more fluid reservoirs; and (c) a pump of the one or more pumps is configured to move fluid from the fluid reservoir to the one or more reagent capsules.
  • Embodiment 4 The system of any one of Embodiments 1-3, wherein (a) the instrument comprises one or more heaters; and (b) at least one of the one or more heaters is configured to interface with the detection region and heat contents thereof.
  • Embodiment 5 The system of any one of Embodiments 1-4, wherein (a) the sample preparation region comprises a lysis region for the lysis one or more components of the sample; and optionally (b) at least one of the one or more reagent capsules comprises lysis reagents.
  • Embodiment 6 The system of Embodiment 4 or 5, wherein at least one of the one or more heaters, is configured to interface with the sample preparation region and heat the contents thereof.
  • Embodiment 7 The system of any one of Embodiments 3-6, wherein (a) the cartridge further comprises a nucleic acid capture region; (b) the one or more pumps comprises an actuator operable to move a sample preparation fluid through the nucleic acid capture region to capture the one or more nucleic acids; and (c) the one or more pumps comprises an actuator operable to move an elution fluid from one of the one or more fluid reservoirs through the nucleic acid capture region to release the captured nucleic acids.
  • Embodiment 8 The system of any one of Embodiments 1-7, wherein at least one of the one or more reagent capsules or the plurality of chambers comprises a programmable nuclease.
  • Embodiment 9 The system of Embodiment 8, wherein (a) the programmable nuclease comprises a Cas protein; and optionally (b) the Cas protein comprises Casl2, Casl3, Casl4, CasPhi, a thermostable Cas, or any combination thereof.
  • Embodiment 10 The system of any one of Embodiments 2-9, wherein (a) the detection reagents further comprise amplification reagents; and optionally (b) the amplification reagents in each chamber of the plurality of chambers comprises one or more primers for amplifying the target nucleic acid bound by the respective targeting sequence.
  • Embodiment 11 The system of any one of Embodiments 2-10, wherein (a) the detection reagents are in a lyophilized form, and/or (b) the guide nucleic acid and/or the reporter in each chamber are immobilized to a surface of the respective chamber.
  • Embodiment 12 The system of any one of Embodiments 1-11, further comprising a gear that translates the cartridge between two or more positions in the instrument.
  • Embodiment 13 The system of Embodiment 12, wherein the two or more positions comprise positions in which (a) one of the one or more pumps aligns with one or more of the plurality of pump interfaces; (b) one of the at least one valve actuator aligns with one or more of the plurality of valves; (c) the optical sensor aligns with the detection region; or (d) any combination thereof.
  • Embodiment 14 The system of any one of Embodiments 4-13, wherein the two or more positions comprise a position in which at least one of the one or more heaters aligns with the sample preparation region or the detection region.
  • Embodiment 15 The system of any one of Embodiments 1-14, further comprising a user interface for receiving instructions from a user.
  • Embodiment 16 The system of any one of Embodiments 1-15, further comprising a non-transitory computer-readable medium with instructions stored thereon, that when executed by one or more processors, (a) activates the one or more pumps and the at least one valve actuator to move fluid between the sample interface, the one or more reagent capsules, the sample preparation region, and the detection region; (b) activates the light source; (c) stores detection results from the optical sensor; and (d) reports results to a user.
  • a non-transitory computer-readable medium with instructions stored thereon that when executed by one or more processors, (a) activates the one or more pumps and the at least one valve actuator to move fluid between the sample interface, the one or more reagent capsules, the sample preparation region, and the detection region; (b) activates the light source; (c) stores detection results from the optical sensor; and (d) reports results to a user.
  • Embodiment 17 A method for detecting one or more of a plurality of different target nucleic acids in a system of any one of Embodiments 1-16, the method comprising:
  • Example 1 Exemplary Workflow - Boom method with silica membrane extraction
  • FIG. 1C The 3-pump, 6-inch cartridge shown in FIG. 1C was designed to execute an implementation of the Boom method for nucleic acid extraction and purification.
  • the functional schematic in FIGS. 2A-2C illustrate the components in the longer, 6-inch cartridge.
  • Six different liquid reagents, 3 pumps, 3 valves, 3 heaters and up to 5 dried reagent chambers are provided in this embodiment to execute the nucleic acid extraction with the Boom method followed by a DETECTR reaction.
  • FIGS. 3-6 highlight the different components that are utilized for each of the steps in the workflow - including lysis, nucleic acid binding to a sample concentrator membrane, washing, elution, and detection.
  • Step 1 - Lysing (FIG. 3):
  • the cells that contain the nucleic acids are lysed in the cartridge. This may be done with heat or a combination of heat and protein degrading enzymes with detergents.
  • lysostaphin or other bacteriocins can be used as lysing agents for specific bacteria that are difficult to lyse.
  • Proteinase K may be used for some assays to digest proteins and enzymes that could interfere with the downstream parts of the molecular diagnostic assay. Heating the specimen with the lysing agents can promote cell degradation.
  • the lysing agent could be all liquid or a combination of liquids in the sample tube and in a liquid reagent capsule.
  • some of the lysing reagents can be dried salts in a dried reagent chamber. This may reduce the chance of the carcinogenic or toxic chaotropic agents from being contacted by the user during the sample collection.
  • Step 2 - Nucleic Acid Binding (FIG. 4): After lysing, the nucleic acids are captured on the surfaces of the sample concentrator (in this embodiment, a silica membrane). A chaotropic substance, such as guanidinium salt, is added to the specimen in during the lysing step to promote a silica surface to adsorb the nucleic acids to the silica surface. For the cartridge, the liquid containing the lysed nucleic acids is pumped through a channel containing a porous silica membrane. The membrane provides a three-dimensional surface area to enable highly efficient nucleic acid binding. The system has a bi-directional pump flow capability to increase the efficiency of binding if a single pass is determined to be insufficient for the assay.
  • a chaotropic substance such as guanidinium salt
  • Step 3 - Washing For many specimen matrixes, especially blood and stool, the cellular and extra-cellular proteins inhibit polymerase amplification reactions.
  • the chaotropic salts used in the lysing and binding steps also inhibit polymerase reactions.
  • the membrane can be washed with one or more reagents to remove proteins and the chaotropic salts to purify the nucleic acids sufficiently for the upcoming molecular polymerase reaction.
  • the chemical constituents of the wash reagents can be different to prepare the bound nucleic acids for Step 4. It may be beneficial to increase purity by having numerous washes. Up to 4 washes can be provided by this particular cartridge design. If fewer washes are required for an assay, one or more reagent capsule silos can be left empty or removed entirely.
  • Step 4 - Elution (FIG. 6):
  • an elution reagent facilitates the nucleic acids release from the silica membrane.
  • the elution reagent is pumped across the silica membrane, and perhaps bi-directionally shuttled back and forth, to increase the efficiency of released nucleic acids. Heat may assist in the elution release.
  • the silica membrane in the cartridge is situated above the instrument’s Heater B.
  • the elution reagent with the nucleic acids is moved through one or more dried reagent chambers into the detection region/DETECTR module.
  • the bi-directional pump capability can be used to fully hydrate the dried reagents and to homogenize in the Pump C transfer/mix chamber with bi-directional flow before entering the DETECTR module.
  • the fluid movement is executed by using Pump C with both Valves B and C.
  • the elution reagent is pumped across the Silica Membrane and through Valve C to the low flow resistance channel with the up to 3 dried reagents.
  • Valve C is switched to closed and Pump C pushes the reagents through the high flow resistance channel and into the DETECTR module.
  • the connection line between valve C and the dried reagent chambers must be kept short to minimize eluted nucleic acid loss.
  • Step 5 - DETECTR reaction (FIG. 6):
  • the programmable nuclease- based detection reaction is passive with respect to fluid movement.
  • the DETECTR module is a one-way flow system. Once the eluted nucleic acids are homogenized with test-agnostic enzymes (that is, programmable nucleases without guides) and other reaction components (such as polymerases, dNTPs, etc.) which were dried in the three dried chambers, Pump C pushes the resulting liquid into the detection region to fill the microwells of the DETECTR module (each containing test-specific reagents such as target-specific primers and guides). A hydrophobic vent in the DETECTR module saturates after the chambers are filled and the liquid flow stops. Pump C stalls, which is detected by its stepper motor encoder, and is stopped.
  • test-agnostic enzymes that is, programmable nucleases without guides
  • other reaction components such as polymerases, d
  • the programmable nuclease-based detection reaction in each microwell utilizes an isothermal polymerase for target amplification (e.g., LAMP, NEAR, SDA, etc.) and a CRISPR-Cas system for a specific signal generation inside each microwell.
  • the DETECTR reaction occurs over instrument’s Heater C as both the polymerase enzyme and the CRISPR enzyme have a thermal range of operation such as 52° C to 65° C depending on the enzyme sets. This thermal range of operation is approximately a log wider in range than what is typical for a PCR reaction.
  • the Boom method may be unnecessary and a lower cost and simpler nucleic acid extraction method can be used.
  • the method can use heat and non-inhibiting lysing reagents.
  • a low pH lysing reagent can be used for the nucleic acid extraction. The low pH is then adjusted with another salt before the detection reaction begins.
  • the simple extraction method can be implemented in the 4.2-inch cartridge shown in FIG. 1A.
  • liquid reagent In this embodiment, shown in FIG. 7, only one liquid reagent is provided for the simple extraction method. It could be housed in the tube used to collect/contain the specimen sample. It also could be in a liquid reagent capsule and mixed with a liquid that stabilizes the specimen when the specimen tube is connected to the cartridge and the cartridge inserted into the instrument.
  • dried reagents provide lysing components, such as to lower the pH, and neutralizing components, such as to restore the pH to a range for the molecular polymerase reaction.
  • the remaining method steps amplification/detection reagent re-hydration, amplification/detection, etc.
  • FIG. 41 for a general description of the lysis, neutralization, activation, and detection steps which could be implemented using a cartridge according to the workflow shown in FIG. 7.
  • Example 3 Designs of Improved Reporters for Immobilization on Substrates
  • a reporter is attached to a surface via any of the chemistries described herein (e.g., amine chemistry).
  • the immobilized reporter then can be contacted with a programmable nuclease and a guide nucleic acid in a trans cleavage reaction.
  • FIGS. 40A-40E show the designs of various immobilized reporters. The arrow below each reporter indicates the expected signal change when the reporter is cleaved by a programmable nuclease.
  • cleavage of the reporter may release a fluorophore, as shown in FIGS. 40A, 40C, and 40E, thereby decreasing the fluorescence signal of the reporter.
  • cleavage of the reporter may release a quencher moiety, thereby increasing the fluorescent signal of the reporter.
  • FIGS. 40A e.g., reporter 112 and 40B (e.g., reporter 136) depict single-stranded nucleic acid reporters that will show decreased and increased fluorescence, respectively.
  • FIGS. 40C e.g., reporter 204) and 40D (e.g., reporter 203) depict two exemplary optimized nucleic acid reporters that will show decreased and increased fluorescence, respectively.
  • FIG. 40E shows a single-stranded nucleic acid reporter with an increased length relative to those of FIGS. 40A-40D.
  • the increased length and/or double-stranded in region in the reporters of FIGS. 40C-40E, relative to those of FIGS. 40A and 40B decrease the steric hindrance of the access of the cleavage sites on the reporter by programmable nuclease.
  • the increased length also decreases interference of access to the cleavage site by the programmable nuclease caused, at least in part, by the surface chemistry of the substrate.
  • FIGS. 33A-33B Exemplary arrangements of chambers connected in sequence in a microfluidic device are shown in FIGS. 33A-33B.
  • the system includes a sample interface configured to receive a sample, wherein the sample interface is in fluid communication with the plurality of chambers. A liquid comprising one or more of the different target nucleic acids or amplicons thereof flows into the plurality of chambers.
  • a complex comprising a programmable nuclease and a guide nucleic acid is activated upon binding a target nucleic acid, and the activated complex cleaves the reporters to form detectable cleavage products in one or more of the wells.
  • the detectable cleavage products are detected, and the location of a well containing the detectable cleavage product identifies the target nucleic acid or amplicon thereof present in the well.
  • the system can include an amplification region in fluid communication with the sample reservoir, and a detection region in fluid communication with the amplification region. A portion of each well fills with fluid before the next sequential well begins to fill with fluid.
  • a guide nucleic acid of known sequence is provided in each well. For reporters comprising a fluorescent label and a quencher, cleavage results in a detectable fluorescence in response to activation at an excitation wavelength.
  • the arrows within the chamber indicate fluid movement.
  • the outlet ports go through to the bottom of the chip where they are covered with a hydrophobic membrane that allows air to pass through but not liquid (except at higher pressures). Due to the fluid movement being laminar at this scale, the movement is driven mainly by surface tension.
  • the main channel is, in this example, 0.40 mm x 0.35 mm in width and depth.
  • the capillary valves are oriented such that they branch off of the main channel at a minimum of 90°.
  • the capillary valve dimensions in this example are 0.30 mm x 0.20 mm in width and depth. The exact dimensions are not required, but in general, the capillary valve inlet area is much smaller than that of the main channel.
  • each well is ⁇ 10uL.
  • the design minimizes fluidic crosstalk between each well. If the fluid volume input to the chip is such that the volume is equal to the amount of volume in each well, air clears the plenum behind each well. If pressure is held, the fluid in each well does not come out of the well and interfere with the reactions occurring in a separate well.
  • the various wells may be arranged in a microfluidic device as illustrated in FIGS. 34A-34C, in which exits extending from the wells to their respective outlets may be of alternating long and short lengths.
  • the various features of the microfluidic device may have a width, depth, area, and ratio as provided in Table 4.
  • the arrows within the chamber indicate fluid movement.
  • the outlet ports are formed at the bottom of the wells.
  • the bottom of the microfluidic device is covered with a hydrophobic membrane that allows air to pass through but not liquid (except at higher pressures). Due to the fluid movement being laminar at this scale, the movement is driven mainly by surface tension.
  • the main channel is, in this example, 0.50 mm x 0.35 mm in width and depth.
  • Each well is roughly 0.50 mm in diameter.
  • the capillary valves are oriented such that they are directly over each well.
  • the capillary valve dimensions in this example are 0.30 mm x 0.20 mm in width and depth.
  • each well is -0.75 pL.
  • the design minimizes fluidic crosstalk between each well, and reduces the fluid volume needed for analysis. If the fluid volume input to the chip is such that the volume is equal to the amount of volume in each well, air clears the plenum behind each well. If pressure is held, the fluid in each well does not come out of the well and interfere with the reactions occurring in a separate well.
  • a sample is inserted into the system, followed by optional lysis and amplification steps.
  • the detection of the cleavage product is achieved by surface characteristics of fluorescence quenching.
  • Each of the programmable nuclease probes contains both a programmable nuclease and a guide nucleic acid.
  • the programmable nuclease at each detection is coupled to a guide nucleic acid that is specific toward a particular target nucleic acid sequence.
  • a different guide nucleic acid resides in each chamber/well. If the desired target is present, an enzyme will be activated and resultant fluorescence/quenching will be observed.
  • the programmable nuclease When the guide nucleic acid of the programmable nuclease selectively binds to its complementary target nucleic acid, the programmable nuclease is activated, thereby enabling trans-cleavage of a cleavable nucleic acid of the reporter.
  • the reporter When the reporter is cleaved, a portion of the reporter is released from the surface (in this example, a quencher). Once the quencher is released, a fluorophore still coupled to the immobilized portion of the reporter is no longer quenched and exhibits fluorescence upon excitation by an illumination source of the instrument. The excitation light from the illumination source in the instrument is transmitted to an optical sensor or detector in the instrument.
  • each chamber/well of the microfluidic device is assessed and any detection event exhibiting fluorescence signifies that the quencher has been released by an activated programmable nuclease, thus indicating that the target nucleic acid specific to the guide nucleic acid of known sequence in the corresponding chamber is present in the sample.
  • the chambers are spatially encoded with known different programmable nuclease-guide complexes, thereby allowing for multiplexed target analysis on a single sample.

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Abstract

La présente invention concerne divers systèmes, dispositifs de diagnostic et procédés d'analyse d'acides nucléiques. Les systèmes, les dispositifs et les procédés permettent l'analyse d'acides nucléiques, dans un échantillon, par l'intermédiaire de dosages à base de nucléases programmables. L'invention concerne des systèmes comprenant un instrument et une cartouche permettant une utilisation au point d'intervention. Les systèmes, les dispositifs et les procédés décrits ici peuvent être conçus pour la détection multiplexée d'acides nucléiques dans un seul échantillon.
PCT/US2024/040578 2023-08-04 2024-08-01 Instrumentation et procédés d'analyse d'acides nucléiques Pending WO2025034518A1 (fr)

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US20160129437A1 (en) * 2014-11-11 2016-05-12 Advanced Liquid Logic, Inc. Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation
US20190264276A1 (en) * 2014-03-11 2019-08-29 Illumina, Inc. Disposable, integrated microfluidic cartridge and methods of making and using same
US20210220829A1 (en) * 2008-09-23 2021-07-22 Bio-Rad Laboratories, Inc. Droplet-based assay system
US20210276008A1 (en) * 2006-03-24 2021-09-09 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
WO2021243308A2 (fr) * 2020-05-29 2021-12-02 Mammoth Biosciences, Inc. Dispositif de diagnostic à nucléase programmable

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
US20210276008A1 (en) * 2006-03-24 2021-09-09 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
US20210220829A1 (en) * 2008-09-23 2021-07-22 Bio-Rad Laboratories, Inc. Droplet-based assay system
US20190264276A1 (en) * 2014-03-11 2019-08-29 Illumina, Inc. Disposable, integrated microfluidic cartridge and methods of making and using same
US20160129437A1 (en) * 2014-11-11 2016-05-12 Advanced Liquid Logic, Inc. Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation
WO2021243308A2 (fr) * 2020-05-29 2021-12-02 Mammoth Biosciences, Inc. Dispositif de diagnostic à nucléase programmable

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